Collections of compounds

ABSTRACT

A compound of formula (I), wherein: R 2  and R 3  are independently selected from H, R, OH, OR, ═O, ═CH—R, ═CH 2 , CH 2 —CO 2 R, CH 2 —CO 2 H, CH 2 —SO 2 R, O—SO 2 R, CO 2 R, COR and CN, and there is optionally a double bond between C1 and C2 or C2 and C3; R 6 , R 7 , R 8  and R 9  are independently selected from H, R, OH, OR, halo, nitro, amino, Me 3 Sn; R 11  is either H or R; Q is S, O or NH; L is a linking group, or a single bond; O is a solid support; or where one or more of R 2 , R 3 , R 6 , R 7  and R 8  are independently: H—(T) n —X—Y—A— where: X is CO, NH, S or O; T is a combinatorial unit; Y is a divalent group such that HY═R; A is O, S, NH, or a single bond and n is a positive integer.

[0001] This invention relates to pyrrolobenzodiazepines, to methods ofsynthesizing these compounds on solid supports, and to collections ofthese compounds. This invention further relates to methods foridentifying and isolating pyrrolobenzodiazepine compounds with usefuland diverse activities from such collections.

BACKGROUND TO THE INVENTION

[0002] Compounds having biological activity can be identified byscreening diverse collections of compounds (i.e. libraries of compounds)produced through synthetic chemical techniques. Such screening methodsinclude methods wherein the library comprises a plurality of compoundssynthesized at specific locations on the surface of a solid supportwhereby a receptor is appropriately labelled to bind to and identify acompound, e.g., fluorescent or radioactive labels. Correlation of thelabelled receptor bound to the support and its location ox the supportidentifies the binding compound (U.S. Pat. No. 5,143,854).

[0003] Central to these methods is the screening of a multiplicity ofcompounds in the library and the ability to identify the structures ofthe compounds which have a requisite biological activity. In order tofacilitate synthesis and identification, the compounds in the libraryare typically formed on solid supports. Usually each such compound iscovalently attached to the support via a cleavable or non-cleavablelinking arm. The libraries of compounds can be screened either on thesolid support or as cleaved products to identify compounds having goodbiological activity.

[0004] A particular class of compounds which would be useful forinclusion in screening libraries are pyrrolobenzodiazepines (PBDs). PBDshave the ability to recognise and bond to specific sequences of DNA; themost preferred sequence is PuGPu (Purine-Guanine-Purine). The first PBDantitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruberet al., 1965 J. Am. Chem. Soc., 87, 5793-5795; Leimgruber et al., 1965J. Am. Chem. Soc., 87, 5791-5793). Since then, a number of naturallyoccurring PBDs have been reported, and over 10 synthetic routes havebeen developed to a variety of analogues (Thurston et al., 1994 Chem.Rev. 1994, 433-465). Family members include abbeymycin (Hochlowski etal., 1987 J. Antibiotics, 40, 145-148), chicamycin (Konishi et al., 1984J. Antibiotics, 37, 200-206), DC-81 (Japanese Patent 58-180 487;Thurston et al., 1990, Chem. Brit., 26, 767-772; Bose et al., 1992Tetrahedron, 48, 751-758), mazethramycin (Kuminoto et al., 1980 J.Antibiotics, 33, 665-667), neothramycins A and B (Takeuchi et al., 1976J. Antibiotics, 29, 93-96), porothramycin (Tsunakawa et al., 1988 J.Antibiotics, 41, 1366-1373), prothracarcin (Shimizu et al, 1982 J.Antibiotics, 29, 2492-2503; Langley and Thurston, 1987 J. Org. Chem.,52, 91-97), sibanomicin (DC-102)(Hara et al., 1988 J. Antibiotics, 41,702-704; Itoh et al., 1988 J. Antibiotics, 41, 1281-1284), sibiromycin(Leber et al, 1988 J. Am Chem. Soc., 110, 2992-2993) and tomamycin(Arima et al., 1972 J. Antibiotics, 25, 437-444).

[0005] PBDs are of the general structure:

[0006] They differ in the number, type and position of substituents, inboth their aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. There is either an imine (N═C), acarbinolamine (NH—CH(OH)) or a carbinolamine methyl ether (NH—CH(OMe))atthe N10-C11 position which is the electrophilic centre responsible foralkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, 1975 in Antibiotics III. Springer-Verlag, New York, pp.3-11; Hurley and Needham-VanDevanter, 1986 Acc. Chem. Res., 19,230-237). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

DISCLOSURE OF THE INVENTION

[0007] A first aspect of the present invention relates to compounds offormula (I):

[0008] wherein:

[0009] R₂ and R₃ are independently selected from: H, R, OH, OR, ═O,═CH—R, ═CH₂, CH₂—CO₂R, CH₂—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR and CN, andthere is optionally a double bond between C1 and C2 or C2 and C3;

[0010] R₆, R₇, R₈ and R₉ are independently selected from H, R, OH, OR,halo, nitro, amino, Me₃Sn; or R₇ and R₈ together from a group—O—(CH₂)_(p)—O—, where p is 1 or 2;

[0011] R₁₂ is either H or R;

[0012] Q is S, O or NH;

[0013] L is a linking group, or less preferably a single bond;

[0014] O is a solid support;

[0015] where R is a lower alkyl group having 1 to 10 carbon atoms, or analkaryl group (i.e. an alkyl group with one or more aryl substituents)preferably of up to 12 carbon atoms, whereof the alkyl group optionallycontains one or more carbon-carbon system, or an aryl group, preferablyof up to 12 carbon atoms; and is optionally substituted by one or morehalo, hydroxy, amino, or nitro groups, and optionally contains one ormore hetero atoms, which may form part of, or be, a functional group;and

[0016] where one or more of R₂, R₃, R₆, R₇ and R₈ may alternatively beindependently X—Y—A—, where X is selected from —COZ′, NHZ, SH, or OH,where Z is either H or a nitrogen protecting group, Z′ is either OH oran acid protecting group, Y is a divalent group such that HY═R, and A isO, S, NH, or a single bond.

[0017] If R is an aryl group and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of, or be, a functional group,e.g. carbonyl, hydroxy, cyano, ester.

[0018] R and HY groups are preferably independently selected from alower alkyl group having 1 to 10 carbon atoms, or an aralkyl group,preferably of up to 12 carbon atoms, or an aryl group, preferably of upto 12 carbon atoms, optionally substituted by one or more halo, hydroxy,amino, or nitro groups. It is more preferred that R and HY groups areindependently selected from a lower alkyl group having 1 to 10 carbonatoms optionally substituted by one or more halo, hydroxy, amino, ornitro groups. It is particularly preferred that R or HY areunsubstituted straight or branched chain alkyl groups, having 1 to 10,preferably 1 to 6, and more preferably 1 to 4, carbon atoms, e.g.methyl, ethyl, propyl, butyl. R may be selected only from methyl andethyl.

[0019] Alternatively, R₆, R₇, R₈ and R₉ may preferably be independentlyselected from R groups with the following structural characteristics:

[0020] (i) an optionally substituted phenyl group;

[0021] (ii) an optionally substituted ethenyl group;

[0022] (iii) an ethenyl group conjugated to an electron sink.

[0023] The term ‘electron sink’ means a moiety-covalently attached to acompound which is capable of reducing electron density in other parts ofthe compound. Examples of electron sinks include cyano, carbonyl andester groups.

[0024] The term ‘nitrogen protecting group’ has the meaning usual insynthetic chemistry, particularly synthetic peptide chemistry. It meansany group which may be covalently bound to the nitrogen atom of anygrouping of the molecule, particularly of the amine grouping, andpermits reactions to be carried out upon the molecule containing thisprotected grouping without its removal. Nevertheless, it is able to beremoved from the nitrogen atom without affecting the remainder of themolecule. Suitable amine protecting groups for the present inventioninclude Fmoc (9-fluorenylmethoxycarbonyl), Nvoc(6-nitroveratryloxycarbonyl), Teoc (2-trimethylsilylethyloxycarbonyl),Troc (2,2,2-trichloroethyloxycarbonyl), Boc (t-butyloxycarbonyl), CBZ(benzyloxycarbonyl), Alloc (allyloxycarbonyl) and Psec(2(-phenylsulphonyl)ethyloxycarbonyl). Other suitable groups aredescribed in Protective Groups in Organic Synthesis, T Green and P Wuts,published by Wiley, 1991 which isincorporated herein by reference.

[0025] The term ‘acid protecting group’ has the meaning usual insynthetic chemistry. It means any group which may be reacted with anycarboxylic acid moiety of the molecule, and permits reactions to becarried out upon the molecule containing this protected grouping withoutits removal. Nevertheless, the carboxylic acid moiety is able to beregenerated without affecting the remainder of the molecule. Suitableacid protecting groups include esters, for example methyl ester, and—O—CH₂═CH₂. Other suitable groups are described in Protective Groups inOrganic Synthesis, T Green and P Wuts, published by Wiley, 1991.

[0026] It is preferred that in compounds of formula I, if one of R₂, R₃,R₆, R₇ and R₈ is to be X—Y—A—, then it is either R₂ or R₈ that isX—Y—A—, and more preferably it is R₈ that is X—Y—A—.

[0027] In compounds of formula I, Q is preferably O, and R₁₁ ispreferably H, Me or ET, more preferably H or Me. Independently, R₆ ispreferably H or R, more preferably H or Me, R₉ is preferably H, and R₇is preferably an alkoxy group, and more preferably methoxy or ethoxy. Itis further preferred that R₂ and R₃ are H.

[0028] If there is a double bond in the pyrrolo C ring, it is preferablybetween C2 and C3.

[0029] A second aspect of the invention relates to compounds of formulaI as defined in the first aspect of the invention except that one ormore of R₂, R₃, R₆, R₇ and R₈ are independently:

H—(T)_(n)—X′—Y—A—

[0030] where:

[0031] Y and A are as defined in the first aspect of the invention;

[0032] X′ is CO, NH, S or O,;

[0033] T is a combinatorial unit;

[0034] and n is a positive integer.

[0035] In compounds of formula I according to the second aspect, it ispreferred that R₇ and/or R₈ are independently:

H—(T)_(n)—X′—Y—A—

[0036] It is preferred that X′ is either CO or NH. n may preferably befrom 1 to 16, and more preferably from 3 to 14. It is also preferredthat it is R₈ which is H—(T)_(n)—X′—Y—A—.

[0037] A third aspect of the present invention relates to compounds offormula II:

[0038] preferably made from a compound of formula I as described in thefirst or second aspect of the invention by removing the compound offormula II from the solid support by cleaving the linking group L, whereR₂, R₃, R₆, R₇, R₈, and R₉ are as defined in the first or second aspectof the invention.

[0039] A fourth aspect of the present invention is a method of making acompound according to the third aspect of the invention from a compoundof formula I as described in the first or second aspect of the inventionby removing the compound of formula II from the solid support bycleaving the linking group L.

[0040] A fifth aspect of the invention relates to a compound of formulaII as described in the third aspect of the invention for use in a methodof therapy. Conditions which may be treated include gene-based diseases,including neoplastic diseases and, for example Alzheimer's disease, andbacterial, parasitic and viral infections.

[0041] In accordance with this aspect of the present invention, thecompounds provided may be administered to individuals. Administration ispreferably in a “therapeutically effective amount”, this beingsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosages etc., is within the responsibility of generalpractitioners and other medical doctors.

[0042] A compound may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

[0043] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may comprise, inaddition to the active ingredient, i.e. a compound of formula II, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous or intravenous.

[0044] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may comprise a solidcarrier or an adjuvant. Liquid pharmaceutical compositions generallycomprise a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other sadcharide solutions, or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol may be included.Capsules may include a solid carrier such as gelatin.

[0045] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and which has suitable pH, isotonicity and stability. Thoseof relevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection or Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required.

[0046] A sixth aspect of the present invention relates to the use of acompound of formula II as described in the third aspect of the presentinvention in the preparation of a medicament for the treatment of agene-based disease or a bacterial, parasitic or viral infection. Thepreparation of a medicament is described in relation to the fourthaspect of the invention. In further aspects, the invention providesprocesses for preparing compounds according to the first and secondaspects of the present invention.

Solid Support

[0047] The term ‘solid support’ refers to a material having a rigid orsemi-rigid surface which contains or can be derivatized to containreactive functionality which can serve to covalently link a compound tothe surface thereof. Such materials are well known in the art andinclude, by way of example, silicon dioxide supports containing reactiveSi—OH groups, polyacrylamide supports, polystyrene supports,polyethyleneglycol supports, and the like. Such supports will preferablytake the form of small beads, pins/crowns, laminar surfaces, pellets ordisks. Other conventional forms may be used.

Linker Group

[0048] The linking groups preferred for the present application are oneswhich contain at least one covalent bond which can be readily broken byspecific chemical reactions, or other changes (e.g. light or a pHchange), thereby providing for liberation of compounds free from thesolid support. The chemical reactions employed to break the covalentbond are selected so as to be specific for the desired bond breakagethereby preventing unintended reactions occurring elsewhere in themolecule. The linking group is selected relative to the synthesis of thecompounds formed on the solid support so as to prevent prematurecleavage of the compound or its precursors from the solid support aswell as to avoid interference with any of the procedures employed duringsynthesis of the compound on the support.

[0049] Examples of linking groups are set out below (shown as availableform), along with suggested cleavage method(s) for the linking group.These groups are commercially available or have been reported in theliterature. After conversion to the appropriate chloroformate, forexample by reaction with triphosgene in the presence of pyridine, theycan be used to attach to anthranilic acids (for use in providing theprotected A-rings of pyrrolobenzodiazepines) via carbamate linkages.Some resins, e.g. p-nitrophenyl carbonate Wang resin may couple to theanthranilic acids without need for intermediate transformation to thechloroformate.

References

[0050] 1. Holmes, C. P., Jones, D. G., “Reagents for CombinatorialOrganic Synthesis: Development of a New O-Nitrobenzyl Photolabile Linkerfor Solid Phase Synthesis”, J. Org. Chem., 60, 2318-2319 (1995).

[0051] 2. Hauske, J. R., Dorff, P. A., “Solid Phase CBZ ChlorideEquivalent. A New Matrix Specific Linker”, Tetrahedron Letters, 36, 10,1589-1592 (1995).

[0052] 3. Kunz, H., Dombo, B., “Solid Phase Synthesis of Peptide andGlycopeptides on Polymeric Supports with Allylic Anchor groups”, AngewChem Int Ed Engl, 5, 711 (1988).

[0053] 4. Garcia-Echeverria, C., “A Base Labile Handle for Solid PhaseOrganic Chemistry”, Tetrahedron Letters, 38, 52, 8933-8934 (1997).

[0054] 5. (a) Albericio, F., Giralt, E., Eritja, R., TetrahedronLetters, 32, 1515 (1991).

[0055] b) Albericio, F., Robles, J., Fernandez-Forner, Y., Palom, C.,Celma, E., Pedroso, E., Giralt, E., Eritja, R., Peptides 1990, Proc 21stEur. Pept. Symp., S134, (1991).

[0056] 6. Mullen, D. G, Barany, G., “A New Fluoridolyzable AnchoringLinkage for Orthogonal Solid-Phase Peptide Synthesis: Design,Preparation, and Application of the N-(3 or4)-[[4-(Hydroxymethyl)phenoxy]-tert-butylphenylsilyl]phenyl PentanedioicAcid Monoamide (Pbs) Handle”, J. Org. Chem., 53, 5240 (1988).

[0057] 7. Dressman, D. A., et al., Tet. Letts., 37, 937 (1996). Allthese documents are incorporated herein by reference.

Combinatorial Unit

[0058] The term ‘combinatorial unit’ means any monomer unit which can beused to build a chain as shown in a compound of formula I as defined inthe second aspect of the present invention, or a compound of formula II,when derived from a compound of formula I as defined in the secondaspect of the present invention. The chain is usually attached to thePBD core by a joining group through the pro N10 position. Examples ofmolecules suitable for such chain building are found in Schreiber et al.(JACS, 120. 1998, pp.23-29), which is incorporated herein by reference.An important example of a unit is an amino acid residue. Chains may besynthesised by means of amine-protected amino acids. Fmoc protectedamino-acids are available from a number of sources, such as Sigma andNovaBiochem. Both natural and unnatural amino acids can be used, e.g. D-and L-amino acids and heterocyclic amino acids. In particular,heterocyclic amino acids of the type found in the construction ofnetropsin and distamycin are of interest because of theirDNA-recognition properties.

[0059] Amine units can be used to make up peptoids: see Soth, M. J. andNowick, J. S. 1997, Unnatural oligomer libraries, Curr. Opin, Chem.Biol. 1, no. 1: 120-129; Zuckermann et al., 1994, Discovery ofNanomolecular Ligands for 7-Transmembrane G-Protein-Coupled Receptorsfrom a Diverse N-(Substituted)glycine Peptoid Library, Journal ofMedicinal Chemistry 37: 2678-85; Figliozzi, GMR et al., 1996, Synthesisof N-substituted Glycine Peptoid Libraries, Methods in Enzymology, 267:437-47; Simon, R. J. et al., 1992, Peptoids: A Modular Approach to DrugDiscovery, Proc. Natl. Acad. Sci. USA, 89:9367-71; which documents areincorporated herein by reference.

[0060] Other combinatorial units include PNAs: P E Nielsen, et al.,Science, 1991, 254, 1497; M Egholm, et al., Nature, 1993, 365, 566; MEgholm et al., JACS, 1992, 114, 1895; S C Brown, et al., Science, 1994,265., 777; 5. K Saha, et al., JOC, 1993, 58, 7827; oligoureas: KBurgess, et al., 1995, Solid Phase Synthesis of Unnatural BiopolymersContaining Repeating Urea Units. Agnew. Chem. Int. Ed. Engl 34, no.8:907; K Burgess, et al., 1997, Solid Phase Synthesis of Oligoureas;Journal of the American Chemical Society 119: 1556-64; andoligocarbamates: E J Moran et al., 1995, Novel Biopolymers for DrugDiscovery. Biopolymers (Peptide Science); John Wiley and Sons 37:213-19; Cho C Y et. al., 1993, An Unnatural Biopolymer. Science 261:1303-5; Paikoff S F et al., 1996, The Solid Phase Synthesis ofN-Alkylcarbamate Oligomers. Tetrahedron Letters 37, no. 32: 5653-56. Allthese documents are incorporated herein by reference.

[0061] A type of combinatorial unit of particular relevance to thepresent invention is one based on the pyrrolobenzodiazepine structures;these are of general formulae IIIa and IIIb:

[0062] wherein R₃, R₆, R₇, R₉, A and Y are as defined in the firstaspect of the invention, A′ and Y′ are independently selected from thepossible groups for A and Y respectively. In order for suchcombinatorial units to be added to the combinatorial chain, they may beadded in their protected form as shown in general formulae IIIc andIIId:

[0063] where R₃, R₆, R₇, R₉, A, Y, A′ and Y′ are as defined above, Q andR₁₁ are as defined in the first aspect of the invention, and R₁₀ is anitrogen protecting group. It is possible that the combinatorial unitsmay remain in their protected form until the compound has been cleavedfrom the solid support, or until any other components of the compoundhave been deprotected.

[0064] The present invention relates to libraries, or collections, ofcompounds all of which are represented by a single one of the formulae Ior II. The diversity of the compounds in a library may reflect thepresence of compounds differing in the identities of one or more of R₂,R₃, R₆, R₇, R₉, R₁₁ and Q and/or in the identities of the combinatorialunits T (when present) The number of members in the library depends onthe number of variants, and the number of possibilities for eachvariant. For example, if it is the combinatorial units which are varied,and there are 3 combinatorial units, with 3 possibilities for each unit,the library will have 27 compounds. 4 combinatorial units and 5possibilities for each unit gives a library of 625 compounds. If forinstance there is a chain of 5 combinatorial units with 17 possibilitiesfor each unit, the total number of members in the library would be 1.4million. A library may therefore comprise more than 1 000, 5 000, 10000, 100 000 or a million compounds, which may be arranged as describedbelow.

[0065] In the case of free compounds (formula II), the individualcompounds are preferably in discrete volumes of solvents, e.g. in tubesor wells. In the case of bound compounds (formula I) the individualcompounds are preferably bound at discrete locations, e.g. on respectivepins/crowns or beads. The library of compounds may be provided on aplate which is of a suitable size for the library, or may be on a numberof plates of a standard size, e.g. 96 well plates. If the number ofmembers of the library is large, it is preferable that each well on aplate contains a number of related compounds from the library, e.g. from10 to 100. One possibility for this type of grouping of compounds iswhere only a subset of the combinatorial units, or substituents, areknown and the remainder are randomised; this arrangement is useful initerative screening processes(see below). The library may be presentedin other forms that are well-known.

[0066] A further aspect of the present invention is a method ofpreparing a collection, or library of compounds as discussed above. Ifthe diversity of the library is in the combinatorial units, then thelibrary may be synthesised by the stepwise addition of protectedcombinatorial units to a PBD core, each step being interposed by adeprotection step. Such a method is exemplified later. If the diversityof the library is in the substituent groups, the library may besynthesised by carrying out the same synthetic methods on a variety ofstarting materials or key intermediates, which already possess-thenecessary. substituent patterns.

[0067] The present invention also relates to a method of screening thecompounds of formula II to discover biologically active compounds. Thescreening can be to assess the binding interaction with nucleic acids,e.g. DNA or RNA, or proteins, or to assess the affect of the compoundsagainst protein-protein or nucleic acid-protein interactions, e.g.transcription factor DP-1 with E2F-1, or estrogen response element (ERE)with human estrogen receptor (a 66 kd protein which functions as ahormone-activated transcription factor, the sequence of which ispublished in the art and is generally available). The screening can becarried out by bringing the target macromolecules into contact withindividual compounds or the arrays or libraries of individual compoundsdescribed above, and selecting those compounds, or wells with mixturesof compounds, which show the strongest effect.

[0068] This effect may simply be the cytotoxicity of the compounds inquestion against cells or the binding of the compounds to nucleic acids.In the case of protein-protein or nucleic acid-protein interactions, theeffect may be the disruption of the interaction studied.

[0069] Protein-protein interactions can be measured in a number of ways,

[0070] e.g. FRET (fluorescence resonance energy transfer) which involveslabelling one of the proteins with a fluorescent donor moiety and theother with an acceptor which is capable of absorbing the emission fromthe donor; the fluorescence signal of the donor will be altereddepending on the interaction between the two proteins. Another method ofmeasuring protein-protein interactions is by enzymatic labelling, using,for example, horseradish peroxidase.

[0071] The screening process may undergo several iterations by selectingthe most active compounds, or groups of compounds, tested in eachiteration; this is particularly useful when testing arrays of wellswhich include mixtures of related compounds. Furthermore, if the wellscontain compounds for which only a subset of the combinatorial units, orsubstituents, are known, but the rest are randomised, subsequentiterations can be carried out by synthesising compounds possessing theselected known (and successful) combinatorial unit, or substituent,pattern, but with further specified combinatorial units, orsubstituents, replacing the previously randomised combinatorial units,or substituents, adjacent the already known pattern; the remainingcombinatorial units, or substituents, are randomised as in the previousiteration. This iterative method enables the identification of activemembers of large libraries without the need to isolate every member ofthe library.

[0072] A further feature of this aspect is formulation of a selectedcompound or selected compounds with pharmaceutically acceptable carriersor diluents.

[0073] A further aspect of the present invention relates to the use ofcompounds of formula II in target validation. Target validation is thedisruption of an identified DNA sequence to ascertain the function ofthe sequence, and a compound of formula II can be used to selectivelybind an identified sequence, and thus disrupt its function.

[0074] Compounds of formula II can also be used in functional genomicsto ascertain the biological function of individual genes, by blockingthis biological action. This is a further aspect of the invention.

Synthesis Methods

[0075] A key step in a preferred route to compounds of formula I is acyclisation procedure to produce the B-ring, involving generation of analdehyde (or functional equivalent thereof) at what will be the11-position, and attack thereon by the pro-10-nitrogen:

[0076] The “masked aldehyde” —CPQ may be an acetal or thioacetal, whichmay be cyclic, in which case the cyclisation involves unmasking.Alternatively, it may be an alcohol —CHOH, in which case the reactioninvolves oxidation, e.g. by means of TPAP or DMSO (Swern oxidation).

[0077] The masked aldehyde compound can be produced by condensing acorresponding 2-substituted pyrrolidine with a 2-nitrobenzoic acid:

[0078] The nitro group can then be reduced to —NH₂ and reacted with asuitable linking group attached to a solid support, e.g. achloroformate, which thereby links the structure to the solid support.

[0079] A process involving the oxidation-cyclization procedure isillustrated in scheme 1 (an alternative type of cyclisation will bedescribed later with reference to scheme 2).

[0080] If R₁₁ is other than hydrogen, the compound of formula I, may beprepared by direct etherification of the alcohol Ia. Compounds with Q═Scan be prepared by treatment of the corresponding alcohol Ia with R₁₁SH,and a catalyst (usually a Lewis Acid such as Al₂O₃). For compounds whereQ═NH, these can be prepared by reacting an amine, R₁₁NH, e.g. C₃H₇NHwith the corresponding alcohol Ia normally with a catalyst, such as aLewis Acid.

[0081] Exposure of the alcohol B to tetrapropylammonium perruthenate(TPAP)/N-methylmorpholine-oxide (NMO) over A4 sieves results inoxidation accompanied by spontaneous B-ring closure to afford thedesired product. The TPAP/NMO oxidation procedure is found to beparticularly convenient for small scale reactions while the use ofDMSO-based oxidation methods, particularly Swern oxidation, provessuperior for larger scale work (e.g. >1 g).

[0082] The uncyclized alcohol B may be prepared by the reaction of theamino alcohol C, generally in solution, with the linking group Lattached to a solid support D. The linking group is preferablyterminated with a chloroformate or acid chloride functionality. Thisreaction is generally carried out in the presence of a base such aspyridine (preferably 2 equivalents) at a low temperature (e.g. at 0° C.)

[0083] The key amino alcohol C may be prepared by reduction of thecorresponding nitro compound E, by choosing a method which will leavethe rest of the molecule intact. For example, treatment of E with tin(II) chloride in a suitable solvent, e.g. refluxing methanol, generallyaffords, after the removal of the tin salts, the desired product C inhigh yield.

[0084] Exposure of E to hydrazine/Raney nickel avoids the production oftin salts and may result in a higher yield of C, although this method isless compatible with the range of possible C and A-ring substituents.For instance, if there is C-ring unsaturation (either in the ringitself, or in R₂ or R₃), this technique may be unsuitable.

[0085] The nitro compound of formula E may be prepared by coupling theappropriate o-nitrobenzoyl chloride to a compound of formula F, e.g. inthe presence of K₂CO₃ at −25° C. under a N₂ atmosphere. Compounds offormula F can be readily prepared, for example by olefination of theketone derived from L-trans-hydroxy proline. The ketone intermediate canalso be exploited by conversion to the enol triflate for use inpalladium-mediated coupling reactions.

[0086] The o-nitrobenzoyl chloride is synthesised from theo-nitrobenzoic acid (or alkyl ester, after hydrolysis) of formula G,which itself is prepared from the vanillic acid (or alkyl ester)derivative H. Many of these are commercially available and some aredisclosed in Althuis, T. H. and Hess, H. J., J. Medicinal Chem., 20(1),146-266 (1977).

Alternative Cyclisation (Scheme 2)

[0087]

[0088] In scheme 1, the final or penultimate step was an oxidativecyclisation. An alternative route, using thioacetal coupling, is shownin scheme 2. Mercury-mediated unmasking causes cyclisation to thedesired compound. (Ia).

[0089] The thioacetal compound may be prepared as shown in scheme 2: thethioacetal protected C-ring [prepared via a literature method: Langley,D. R. & Thurston, D. E., J. Organic Chemistry, 52, 91-97 (1987)] iscoupled to the o-nitrobenzoic acid (or alkyl ester) G using a literatureprocedure. The resulting nitro compound cannot be reduced byhydrogenation because of the thioacetal group, so the tin(II) chloridemethod is used to afford the amine. This is then N-protected, e.g., byreaction with a chloroformate or acid chloride, such asp-nitrobenzylchloroformate.

[0090] Acetal containing C-rings can be used as an alternative in thistype of route with deprotection including other methods, including theuse of Lewis Acid conditions (see example 3).

[0091] In the above synthesis schemes, the derivatisation of the A-ringis shown as being complete before the compounds are attached to thesolid support. This is preferred if the substituents are groups such asalkoxy or nitro. On the other hand, substituent groups such as alkyl oralkenyl could be added to the A-ring after the coupling of the compoundto the sorid support. This may be achieved by R₆, R₇, R₈ or R₉ beingeasily replaceable groups, such as a halogen atom.

[0092] An alternative synthesis route (as in Examples 3 and 4—FIGS. 4and 5) is to attach the component which will form the A ring to thesolid support at the pro N10 position, before joining the componentwhich will form the C ring.

[0093] Embodiments of the present invention will now be described by wayof example with reference to the accompanying drawings in which:

[0094]FIG. 1 is a synthesis scheme for a compound according to theinvention;

[0095]FIG. 2 is a synthesis scheme for another compound according to theinvention;

[0096]FIG. 3 is a synthesis scheme for an intermediate in the synthesisof a compound according to the invention;

[0097]FIGS. 4 and 5 are synthesis schemes for further compoundsaccording to the invention;

[0098]FIG. 6 is an HPLC time course for cleavage of the compound made bythe scheme shown in FIG. 2;

[0099]FIG. 7 is a graph which illustrates the results shown in FIG. 6;

[0100]FIG. 8 is a graph illustrating the cytotoxicity of the compoundmade by the scheme shown in FIG. 2; and

[0101]FIGS. 9-12 are a synthesis scheme for further compounds accordingto the invention.

General Methods

[0102] Melting points (mp) were determined on a Gallenkamp P1384 digitalmelting point apparatus and are uncorrected. Infrared (IR) spectra wererecorded using a Perkin-Elmer 297 spectrophotometer. ¹H- and ¹³C-NMRspectra were recorded on a Jeol GSX 270 MHZ FT-NMR spectrometeroperating at 20° C.+/−1° C. Chemical shifts are reported in parts permillion (δ) downfield from tetramethylsilane (TMS). Spin multiplicitiesare described as: s (singlet), bs (broad singlet), d (doublet), dd(doublet of doublets), t (triplet), q (quartet), p (pentuplet) or m(multiplet). Mass spectra (MS) were recorded using a Jeol JMS-DX 303 GCMass Spectrometer (EI mode: 70 eV, source 117-147° C.). Accuratemolecular masses (HRMS) were determined by peak matching usingperfluorokerosene (PFK) as an internal mass marker, and FAB mass spectrawere obtained from a glycerol/thioglycerol/trifluoroacetic acid(1:1:0.1) matrix with a source temperature of 180° C. Optical rotationsat the Na-D line were obtained at ambient temperature using aPerkin-Elmer 141 Polarimeter. Analytical results were generally within+/−0.2% of the theoretical values. Flash chromatography was performedusing Aldrich flash chromatography “Silica Gel-60” (E. Merck, 230-400mesh). Thin-layer chromatography (TLC) was performed using GF₂₅₄ silicagel (with fluorescent indicator) on glass plates. All solvents andreagents, unless otherwise stated, were supplied by the Aldrich ChemicalCompany Ltd. and were used as supplied without further purification.Anhydrous solvents were prepared by distillation under a dry nitrogenatmosphere in the presence of an appropriate drying agent, and werestored over 4A molecular sieves or sodium wire. Petroleum ether refersto the fraction boiling at 60-80° C.

Overall Synthetic Stratecy for Examples 1 and 2

[0103] The pyrrolobenzodiazepine products 8 and 13 were obtained insolution by exposure to light at 365 nm; light at this wavelengthpromotes the conversion of the photolabile linker into a nitrosoaldehyde, in the process liberating the PBD from the resin. In additionto this photolabile linker, other fluoride, mild acid, mild base orpalladium (0)/nucleophile labile linkers may also be used in theconstruction of PBD libraries.

[0104] The bead bound PBDs 7 and 12 (FIGS. 1 and 2 respectively) wereprepared by oxidation of the primary-alcohol-bearing resins 6 and 11with SO₃.Pyridine complex in DMSO. Other oxidizing systems such asTPAP/NMO, the Dess Martin reagent, and oxalyl chloride/DMSO (Swernoxidation) are also effective (see example 5). The primary alcoholresins 6 and 11 were obtained from the coupling of the bead boundanthranilic acids 4 and 10 to pyrrolidine methanol 5. Alternatively,coupling (2S, 4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxy proline tothe bead-bound anthranilic acid offers the opportunity of elaboratingthe PBD C-ring at the pro-C2-position on bead via, for example,olefination. Finally, the bead bound anthranilic acids 4 and 10 wereobtained by coupling the commercially available anthranilic acids (over40 anthranilic acids are commercially available) to theo-nitrobenzylchloroformate resin 2 which was in turn obtained from thecommercially available resin 1 by treatment with triphosgene in thepresence of dry pyridine.

EXAMPLE 1 Synthesis of the Resin-Bound C7-Iodo-PBD Carbinolamine (7)(FIG. 1)

[0105] Ortho-Nitro Benzyl Chloroformate Resin (2)

[0106] Hydroxymethyl-photolinker NovaSyn TG resin 1 (0.2 g, 0.24 mmol/gloading) was placed in a vessel, fitted with a sinter. DichloromethaneCH₂Cl₂ (3 mL) was added and the vessel shaken for 30 minutes. Thesuspension was then cooled to 0° C. before adding triphosgene (0.15 g,0.5 mmol) in CH₂Cl₂ and pyridine (40 μL, 0.5 mmol), and the vesselallowed to shake at room temperature for 16 hours. The chloroformateresin 2 was collected by filtration and rinsed with CH₂Cl₂ (2×5 mL) andMeOH (2×5 mL), and dried in vacuo. IR (reflectance, cm⁻¹): 1700 (C═O).

[0107] Attaching Iodinated A-Ring to form Resin (4)

[0108] Dichloromethane CH,CL, (5 mL) was added to resin 2 (0.048 mmol)and the vessel was allowed to shake for 30 minutes. The suspension wasthen cooled to 0° C. and a solution of iodoanthranilic acid 3 (0.13 g,0.48 mmol) and pyridine (40 μl) in NMP (2 mL) was added, and the vesselwas allowed to shake at room temperature for 16 hours. Resin 4 was thencollected by filtration, rinsed with CH₂Cl₂ (2×5 mL), NMP (2×5 mL) andMeOH (2×5 mL), and dried in vacuo. HPLC analysis after release ofiodoanthranilic acid by irradiation indicated that 58% of availablesites had been carbanoylated. IR (reflectance, cm⁻¹): 1750-1650 (CONH).

[0109] Attaching Pyrrolo C-Ring to from Resin (6)

[0110] Dimethyl formamide DMF (5 mL) was added to resin 4 (0.036 mmol)and the vessel allowed to shake for 30 minutes. Pyrrolidine methanol 5(40 μl, 0.36 mmol), TBTU (0.12 g, 0.36 mnmol) in DMF (1 mL) and DIPEA(65 μl, 0.36 mmol) were added, and the vessel allowed to shake at roomtemperature for 16 hours. Resin 6 was collected by filtration, rinsedwith CH₂Cl₂ (2×5 mL) and MeOH (2×5 mL), and dried in vacuo. IR(reflectance, cm⁻¹): 1650-1600 (C═O).

[0111] B-Ring Cyclisation to form Bead-Bound Carbinolamine (7)

[0112] Dichloromethane CH₂Cl₂ (0.5 mL) was added to resin 6 (0.024 mmol)and the vessel allowed to shake for 30 minutes. The suspension was thencooled to −10° C., and triethylamine (10 μl, 0.072 mmol) and sulphurtrioxide.pyridine complex (0.012 g, 0.072 mmol) in DMSO (0.25 mL) added.Shaking was continued for 1 hour at 10° C., and the resin 7 was thencollected by filtration, rinsed with CH₂Cl₂ (2×5 mL) and MeOH (2×5 mL),and dried in vacuo.

[0113] The resulting compound 7 may be cleaved from the solid support byUV light of a wavelength of 365 nm to form a compound of formula 8.

[0114] Further Synthesis Steps

[0115] The compound of formula 7 may serve as a starting point for thesynthesis of a wide variety of other compounds. The iodine at the C8position can be reacted with a boronic acid with Pd (PPh₃), as acatalyst in modified Suzuki reaction. An alternative synthesis route isto stanylate the C8 position by reacting the compound of formula 8 withMe₆Sn₂, with Pd (PPh₃)₄ as a catalyst. The stanylated compound iscapable of coupling with acrylates (i.e. the Heck reaction), iodo- andbromo-arenes (i.e. the Suzuki reaction) and haloalkenes (i.e. the Stillereaction).

EXAMPLE 2 Synthesis of Resin-bound 7,8-Dimethoxy PBD (12) (FIG. 2)

[0116] Attaching Dimethoxy A-Ring to form Resin (10)

[0117] Dichloromethane CH₂Cl₂ (2 mL) was added to the choloroformateresin 2 (0.05 mmol) (prepared as in Example 1) and the vessel allowed toshake for 30 minutes. The suspension was cooled to 0° C., a solution of4,5-dimethoxyanthranilic acid 9 (0.05 g, 0.25 mmol) and pyridine (20 μL)in NMP (2 mL) added, and the vessel allowed to shake at room temperaturefor 16 hours. The resin 10 was collected by filtration, and then rinsedwith CH₂Cl₂ (2×5 mL), NMP (2×5 mL) and MeOH (2×5 mL). The entireprocedure was repeated twice and the resin was then dried in vacuo. IR(reflectance, cm⁻¹): 1750-1650 (CONH).

[0118] Attaching Pyrrolo C-Ring to form Resin (11)

[0119] Dimethyl formamide DMF (5 mL) was added to resin 10 (0.05 mmol)and the vessel allowed to shake for 30 minutes. Pyrrolidine methanol 5(0.025 g, 0.25 mmol), TBTU (0.08 g, 0.25 mmol) in DMF (1 mL) and DIPEA(45 μL, 0.25 mmol) were added, and the vessel allowed to shake at roomtemperature for 16 hours. The resin 11 was collected by filtration, andrinsed with DMF (2×5 mL), NMP (2×5 mL) and CH₂Cl₂ (2×5 mL). The entireprocedure was repeated twice, and the resin then dried in vacuo. IR(reflectance, cm⁻¹): 1700-1600 (C═O).

[0120] B-Ring Cyclisation to form Bead-Bound Carbinolamine (12)

[0121] Dichloromethane CH₂Cl₂ (1 mL) was added to resin 11 (0.05 mmol)and the vessel allowed to shake for 30 minutes. The suspension wascooled to −10° C., and triethylamine (20 μL, 0.15 mmol) and sulphurtrioxide.pyridine complex (0.024 g, 0.15 mmol) in DMSO (0.5 mL) wereadded. The suspension was then allowed to warm to room temperature, andthe vessel was left to shake for 2 hours. The resin 12 was collected byfiltration and rinsed with CH₂Cl₂ (2×5 mL) and MeOH (2×5 mL). The entireprocedure was repeated twice and the resin then dried in vacuo.

EXAMPLE 3 Alternative Synthesis of Resin-bound 7,8-Dimethoxy PBD (21)(FIGS. 3 & 4)

[0122] Overall Synthetic Strategy

[0123] The on-bead oxidation step employed in the previous approachescan be avoided by coupling an anthranilic acid loaded resin to thedimethyl acetal 16 derived from proline (FIG. 4). In this approach,unmasking of the dimethyl acetal protected aldehyde leads to spontaneousB-ring closure. Thus, exposure of the acetal 20 to a palladium catalyst(Pd(CH₃CN)₂Cl₂) leads to the formation of the cyclized compound 21. Theacetal 20 was derived from the anthranilic acid resin 19 and the acetal16, which were coupled together under standard conditions. The acetal 16was obtained from the Cbz protected compound 15 (FIG. 3) byhydrogenation; i5 was in turn prepared by acetalisation of the aldehyde14. Swern oxidation of the primary alcohol 13 afforded the aldehyde 14,the primary alcohol was prepared by a lithium tetrahydroborate reductionof the commercially available Cbz protected proline ester ester 12.

[0124] (2S-N-(benzoxycarbonyl)-2-hydroxymethylproline (13)

[0125] Lithium tetrahydroborate (2.6 g, 0.12 mol) was added portionwiseto a solution of N-Carbobenzyloxy-L-proline methyl ester 12 (21 g, 0.08mol) in THF (500 mL) at 0° C. The reaction mixture was allowed to stirat room temperature for 48 hours. The solution was then cooled to 0° C.and ice water (150 mL) was added to quench excess lithiumtetrahydroborate. The resulting suspension was adjusted to pH 4.0 withaqueous HCl (1.0 N) and extracted with Et₂O (250 mL). The organic phasewas separated and washed with H₂O (3×100 mL), brine (2×100 mL), dried(MgSO₄) and concentrated to give alcohol 13 as a pale yellow oil (18.6g, 99%). ¹H NMR (270 MH_(z), CDCl₃) δ 2.1-1.77 (m, 4H); 3.76-3.35 (m,4H); 4.1-3.77 (m, 1H); 5.14 (2×s, 2H); 7.38-7.28 (m, 5H). CIMS 236 (M⁺).

[0126] (2S)-N-benzoxycarbonyl)pyrrolidine-2-carboxaldehyde (14)

[0127] A solution of triethylamine (32 mL, 0.23 mol) and SO₃.pyridinecomplex (37 g, 0.23 mol) in DMSO (210 mL) a solution of alcohol 13 (18g, 0.077 mol) in CH₂Cl₂ (250 mL) at −10° C., under a nitrogenatmosphere. The reaction mixture was allowed to warn to room temperatureand stirred for 30 minutes and then poured into ice water (200 mL) andextracted with Et₂O. The organic phase was washed with aqueous HCl (1.0N, 3×150 mL), H₂O (3×150 mL), brine (2×150 mL), dried (MgSO₄) andconcentrated to give a yellow oil. The crude material was purified byflash column chromatography (EtOAc) to give aldehyde 14 as a colourlessoil (12.6 g, 71%). ¹H NMR (270 MH_(z), CDCl₃) δ 2.16-1.8 (m, 4H);3.66-3.5 (m, 2H); 4.22-4.17 (m, 1H); 5.22-5.13 (m, 2H); 7.37-7.3 (m,5H); 9.59 (2×s, 1H). CIMS 234 (M⁺+1).

[0128] (2S)-N-(benzoxycarbonyl)pyrrolidine-2-carboxaldehyde dimethylacetal (15)

[0129] Thionyl chloride (5.5 mL) was added to a solution of aldehyde 14(11 g, 0.047 mol) and trimethyl orthoformate (36 mL, 0.33 mol) in MeOH(55 mL) at 0° C. The reaction mixture was heated at 60° C. for 2 hours.The solution was allowed to cool to room temperature, and treated withexcess solid Na₂CO₃ and diluted with Et₂O (60 mL). The suspension wasfiltered to remove insoluble inorganics and resultant filtrate wasconcentrated in vacuo and the redissolved in EtOAc. The organic solutionwas washed with saturated aqueous NaHCO₃ (3×50 mL), brine (2×50 mL),dried (MgSO₄) and concentrated to give the acetal 15 as a yellow liquid(12.5 g, 95%). ¹H NMR (270 MH_(z), CDCl₃) δ 2.16-17 (m, 4H); 3.64-3.33(m, 4.02-3.91 (br. m, 1H); 4.4 and 4.6 (2×br. s, 1H); 4.4 and 4.6 (2×br.s, 1H); 5.17-5.1 (m, 2H); 7.47-7.28 (m, 5H).

[0130] Pyrrolidine-2-carboxaldehyde dimethyl acetal (16)

[0131] A solution of acetal 15 (5.8 g, 0.02 mol) in EtOH (50 mL) wasallowed to stir for 16 hours at room temperature over Raney nickel (0.2g), in order to remove the trace amounts of sulphur impurities prior tohydrogenation. Excess nickel was removed by filtration through Celite.

[0132] 10% palladium on carbon (580 mg) was added to the alcoholicsolution which was subjected to hydrogenation under pressure (c. 50psi). After 16 hours, the reaction mixture was filtered through Celiteand the pad washed with EtOAc, the combined organic solutions wereconcentrated to give the secondary amine 16 as a pale green liquid (2.9g, 100%). ¹H NMR (270 MH_(z), CDCl₃) δ 1.93-1.59 (m, 4H); 3.1-2.92 (m,2H); 3.4-3.3 (d, J=6.9 Hz, 1H); 3.41 (2×s, 6H); 3.53 (br. s, 1H); 4.2(d, J=6.8 Hz, 1H).

[0133] Synthesis of Resin-bound Methyl Ester 21 (FIG. 4)

[0134] A suspension of hydroxyethyl-photolinker NovaSyn TG resin 17(0.114 g, 0.24 mmol/g loading) in CH₂Cl₂ (1 mL) in a vessel fitted witha sinter was shaken for 30 minutes. The suspension was cooled to 0° C.,before addition of a solution of triphosgene (0.04 g, 0.14 mmol) andpyridine (11 mL, 0.14 mmol) in CH₂Cl₂ (0.5 mL) The vessel was allowed toshake at room temperature for 16 hours. The resin 18 was filtered andrinsed with CH₂Cl₂ (2×2 mL) NMP (2×2 mL) and CH₂Cl₂ (2×2 mL). Thisprocedure was repeated twice and the resin was then dried in vacuo.

[0135] A suspension of resin 18 (0.027 mmol) in CH₂Cl₂ (1 mL) wasallowed to shake for 30 mins. The suspension was cooled to 0° C. and asolution of 4,5-dimethoxy-anthranilic acid 8 (0.03 g, 0.14 mmol) andpyridine (10 mL) in NMP (0.5 mL) was added. The vessel was allowed toshake at room temperature for 16 hours.

[0136] Resin 19 was filtered and rinsed with CH₂Cl₂ (2×2 mL), NMP (2×2mL) and MeOH (2×2 mL). The procedure was repeated twice and then theresin was dried in vacuo.

[0137] A suspension of resin 19 (0.027 mmol) in DMF (1 mL) was allowedto shake for 30 minutes. To this suspension was added the acetal 16 (20mg, 0.14 mmol), TBTU (43 mg, 0.14 mmol) and DIPEA (25 mL, 0.144 mmol) inDMF (0.5 mL). The vessel was allowed to shake at room temperature for 2hours after which time the resin 20 was filtered and rinsed with DMF(2×1 mL), CH₂Cl₂ (2×1 mL) and MeOH (2×1 mL). The procedure was repeatedtwice and then the resin was dried in vacuo.

[0138] A suspension of resin 20 (0.027 mmol) in acetone (0.5 mL) wasallowed to shake for 30 minutes. To this suspension was addedPdCl₂(CH₃CN)₂ (7 mg, 0. 027 mmol) in acetone (0. 4 mL) and the vesselwas allowed to shake at room temperature for 2 hours. The resultingresin 21 was filtered and rinsed with acetone (2×1 mL), CH₂Cl₂ (2×1 mL)and MeOH (2×1 mL). The procedure was repeated twice and then the resinwas dried in vacuo

EXAMPLE 4 Further Alternative Synthesis of 7,8-Dimethoxy PBD (13) (FIG.5)

[0139] Overall Synthetic Strategy

[0140] This synthesis used the on-bead oxidation step of example 1 and 2to obtain the resin-bound PBD 34, but using the Dess Martin reagent andSwern oxidation. The resin used to bind the required anthranilic acid 31is p-nitrophenyl carbonate Wang resin, which can directly couple theanthranilic acid without the need for interactive transformation to thechloroformate and thus eliminating a process step.

[0141] Synthesis Route

[0142] A suspension of p-nitrophenyl carbamate Wang resin 31 (1 g, 0.93mmol/g loading) in CH₂Cl₂/DMF (2:1, 10 mL) was shaken for 30 minutes. Asolution of dimethoxyanthranilic acid 9 (0.92 g, 4.7 mmol), HOBt (0.37g, 2.8 mmol) and DIPEA (0.97 mL, 5.5 mmol) in CH₂Cl₂/DMF (2:1, 20 mL)was added to the swollen resin. The vessel was allowed to shake at roomtemperature for 6 hours. Resin 32 was filtered and rinsed with DMF (2×10mL), CH₂Cl₂(2×10 mL), MeOH (2×10 mL), Et₂O (10 mL) and dried in vacuo.

[0143] A suspension of resin 32 (0.93 mmol) in DMF (10 mL) was allowedto shake for 30 minutes. A solution of pyrrolidine methanol 5 (0.47 g,4.7 mmol), TBTU (1.5 g, 4.7 mmol) and DIPEA (0.81 mL, 4.7 mmol) in DMF(10 mL) was added to the swollen resin. The vessel was allowed to shakeat room temperature for 6 hours. Resin 33 was filtered and rinsed withDMF (2×10 mL), CH₂Cl₂ (2×10 mL), MeOH (2×10 mL), Et₂O (10 mL) and driedin vacuo. This entire procedure was repeated once.

[0144] A suspension of resin 33 (0.93 mmol) in CH₂Cl₂ (10 mL) wasallowed to shake for 30 min. A solution of Dess Martin periodinane (1.97g, 4.7 mmol) in CH₂Cl₂ (20 mL) was added to the swollen resin. Thevessel was allowed to shake at room temperature for 2 hours. Resin 34was filtered and rinsed with CH₂Cl₂ (2×10 mL), MeOH (2×10 mL), Et₂O (10mL) and dried in vacuo.

[0145] A suspension of resin 34 (0.93 mmol) in TFA/CH₂Cl₂ (20 mL) wasallowed to shake for 2 hours. The resultant red solution was decantedoff and the procedure was repeated on the remaining resin, to ensurecomplete cleavage. The combined organic solution was diluted with water(20 mL) and carefully neutralised to pH 7.0 by the addition of solidsodium bicarbonate. The organic phase was separated and washed with H₂O(3×20 mL), brine (2×20 mL), dried (MgSO₄) and concentrated to give a redfilm. The crude material was purified by flash column chromatography(silica gel, 1% MeOH/CHCl₃) to give imine 13, as a beige solid (142 mg,59%).

[0146]¹H NMR (270 MHz, CDCl₃) δ 2.4-1.26 (m, 6H), 3.9-3.82 (m, 1H), 3.96and 3.93 (2×s, 6H), 6.81 (s, 1H), 7.52 (s, 1H), 7.69-7.67 (d, J=4.2 Hz,1H); ¹³C NMR (68.7 MHz, CDCl₃) δ 24.2, 29.4, 38.7, 46.7, 53.7, 56.4,109.4, 111.3, 120.3, 140.7, 147.5, 151.3, 162.5, 164.6.

[0147] Alternative Oxidation Method:

[0148] A suspension of resin 33 (0.6 mmol) in CH₂Cl₂ (10 mL) was allowedto shake for 30 min. A solution of SO₃-pyridine complex (0.96 g, 6 mmol)and triethylamine (5 mL, 0.036 mol) in DMSO (5 mL) was added to theswollen resin. The vessel was allowed to shake at room temperature for 3hours. Resin 34 was filtered and rinsed with CH₂Cl₂ (2×10 mL), MeOH(2×10 mL), Et₂O (10 mL) and dried in vacuo.

[0149] A suspension of resin 34 (0.6 mmol) in TFA/CH₂Cl₂ (1:1, 20 mL)was allowed to shake for 3 hours. The resultant red solution wasdecanted off and was diluted with water (20 mL) and carefullyneutralised to pH 7.0 by the addition of solid sodium bicarbonate. Theorganic phase was separated and washed with H₂O (3×20 mL), brine (2×20mL), dried (MgSO₄) and concentrated to give a luminous yellow oil. Thecrude material was purified by flash column chromatography (silica gel,5% MeOH/CHCl₃) to give imine 13, as a yellow film (60 mg, 38%) (NHR asabove).

EXAMPLE 5 Synthesis of Three Resin Bound PBDs (52, 53, 54)

[0150]

[0151] Compounds 52 and 53 were synthesised in the same way as example4, using the alternative oxidation method (Swern) starting from theappropriate anthranilic acids (52: 5-methoxyanthranilic acid; 53:5-methylanthranailic acid). Compound 54 was synthesieed in the same wayas example 4 using the first oxidation method (Dess Martin) startingfrom 6-methylanthranilic acid. The EIMS (M+H)⁺ results for thecompounds, after cleavage from the solid support, were: 52-230; 53-214;54-215.

EXAMPLE 6 Cleavage of PBDs from Beads

[0152] HPLC Method

[0153] Assays of the PBDs synthesised in example 2 were carried out on areversed-phase 25 cm×4.6 mm (inside diameter) C4 (Nucleosil™; 5 μm beadsize) column protected with a Delta-Pak™ C.4, 300 Å Guard pre-column.Elution was carried out using a mobile phase consisting of MeOH/H₂O(1:1) at a flow rate of 1 mL/min. A Waters 490E multiwavelength detectorwas used. Peak identification was accomplished by reference to anauthentic sample of compound 13 synthesized “off-bead”. ConditionsINJECTION VOLUME: 20 μL FLOW RATE: 1 mL/min MOBILE PHASE: 50%METHANOL/50% WATER STATIONARY PHASE: C4, 5 μm (REVERSED PHASE) COLUMN:WATERS 300 Å DETECTOR: 254 nm RUN TIME: 20 mins

[0154] Cleavage of the PBD from beads following UVA irradiation wasmonitored by HPLC. The resin-bound compound 12 (JMB 98) at aconcentration of 1 mM in DMF was UVA irradiated. At appropriate timeintervals, samples were centrifuged to pellet the beads and the amountof free PBD released into the supernatant determined by HPLC. Afterphotolysis of resin 12, carbinolamine 13 was the only species producedas determined by reference to an authentic sample of compound 13synthesised “off bead”. Typical HPLC traces of authentic 13 and of thePBD cleaved from resin 12 with increasing irradiation times are shown inFIG. 6, and the percentage cleavage with time shown in FIG. 7. Cleavageoccured linearly with time, and complete cleavage was achieved by 2hours under the conditions used. HPLC studies indicated that 77% of thesites on the beads had reacted.

In Vitro Cytotoxicity Assay

[0155] MTT Assay Method

[0156] The ability of agents to inhibit the growth of chronic humanhistiocytic leukaemia U937 cells or human chronic myeloid leukaemia K562cells in culture was measured using the MTT assay (Mosmann, 1983). Thisassay is based on the ability of viable cells to reduce a yellow solubletetrazolium salt, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma Chemical Co.), to an insoluble purple formazanprecipitate. Cells at a density of 5×104 cells/mL were continuouslyincubated with the test compounds at a final concentration of 0.3 μM.Aliquots of each of the compounds of the 27-member library were eitherleft without UVA (365 nm) exposure or were exposed to UVA (365 nm) for 2hours prior to their addition to the cell suspension. Following drugtreatment, the cells were transferred to 96-well microtitre plates, 10⁴cells per well, 8 wells per sample. The plates were incubated at 37° C.in a humidified atmosphere containing 5% CO₂. Following incubation ofthe plates for 4 days (to allow control cells to increase in number10-fold), 20 μL of a 5 mg/mL solution of MTT in phosphate-bufferedsaline was added to each well and the plates further incubated for 5hours. The plates were then centrifuged for 5 minutes at 300 g, and thebulk of the medium removed from the cell pellet, leaving 10-20 μL perwell. DMSO (200 μL) was added to each well, and the samples agitated toensure complete mixing. The optical density was then read at awavelength of 550 nm using a Titertek Multiscan ELISA plate reader andthe dose-response curve constructed. The IC, value was read as the doserequired to reduce the final optical density to 50% of the controlvalue.

[0157] Results

[0158] The cytotoxicity of the PBD released from resin 12 followingirradiation was determined using the MTT assay. The survival curveresulting from the compound released from 12 (JMB 98) following 2 and 5hours irradiation was consistent with that of authentic 13 (AG 105); seeFIG. 8. The released PBD therefore has full biological activity.

EXAMPLE 7 Synthesis of a Resin-Bound 8-Aminopropyl PBD Scaffold (30)(see FIG. 9)

[0159] Overall Synthetic Strategy

[0160] The o-nitrobenzylchloroformate resin 2 can also immobilize morecomplicated amines other than simple anthranilic acids, greatlyfacilitating the preparation of molecules such as the PBD C8-aminoscaffold 30. As in the previous strategy, the Fmoc protected scaffold 29was prepared by oxidizing the primary alcohol resin 28. This resin wasobtained by loading the o-nitrobenzylchloroformate resin 2 with theamino alcohol 27. The amino alcohol was prepared by a Tin (II) chloridemediated reduction of the nitro alcohol 26; use of hydrogenationconditions to reduce the nitro group were avoided due to the presence ofthe Fmoc group in 26. The nitro alcohol in turn was furnished in thiscase by coupling pyrrolidine methanol 5 to the o-nitrobenzoic acid 25,although other functionalised prolines could also be employed in thecoupling reaction. The Fmoc o-nitrobenzoic acid was obtained via Fmocprotection of the amino acid 24 produced by hydrolysis of the ester 23.Other nitrogen protecting groups may be substituted for Fmoc as long asthe cleavage conditions involved are compatible with the presence of ano-nitrobenzyl carbamate linker (eg. Boc, Alloc, Teoc etc). Finally, theamino ester was prepared by nitration of 22 which was obtained by aMitsunobu etherification of commercially available methyl vanillate.

[0161] Boc Amino Ester (22)

[0162] A solution diethylazidodicarboxylate (3.38 g, 19.4 mmol) in THF(50 mL) was added dropwise to a solution of methylvanillate (3.53 g,19.4 mmol), N-Boc-propanolamine (3.4 g, 19.4 mmol) andtriphenylphosphine (5.09 g, 19.4 mmol) in THF (50 mL) at 0° C. Thereaction mixture was allowed to warm to room temperature and stirovernight. Excess solvent was removed by rotary evaporation underreduced pressure and the residue triturated with toluene. Precipitatedtriphenylphosphine oxide was removed by vacuum filtration and thefiltrate concentrated in vacuo. The residue was subjected to flashcolumn chromatography (silica gel, petroleum ether 40-60/ethyl acetate,80/20) and removal of excess eluent afforded the pure product 22 (4.8 g,73% yield.). ¹H NMR (270 MHz, CDCl₃) δ 7.65 (dd, J=8.43, 2.02 Hz, 1H),7.54 (d, J=2.02 Hz, 1H), 6.86 (d, J=8.43 Hz, 1H), 5.55 (bs, 1H), 4.15(t, J=5.87 Hz, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 3.41-3.35 (m, 2H),2.09-2.00 (m, 2H) and 1.46 (s, 9H). ¹³C NMR (68.7 MHz, CDCl₃) δ 166.9,156.1, 152.1, 148.8, 123.5, 122.8, 112.0, 111.2, 79.0, 68.2, 55.9, 52.0,38.9, 29.2 and 28.5.

[0163] Amino Nitro Ester (23)

[0164] The Boc-protected amine 22 (10 g) was added portionwise to coldnitric acid (30 mL, 70%, ice bath), the reaction mixture was allowedwarm to room temperature and stir overnight. The reaction mixture waspoured onto crushed ice (100 9) and the resulting aqueous solutionreduced to half its original volume by rotary evaporation under reducedpressure. The resulting precipitate was collected by vacuum filtrationand recrystallised from absolute ethanol to afford the product as ayellow crystalline solid 23 (8.9 g, 87%). ¹H NMR (270 MHz, CDCl₃) δ 7.47(s, 1H), 7.08 (s, 1H), 4.24 (t, J=5.86 Hz, 2H), 3.96, (s, 3H), 3.89 (s,3H), 3.24 (t, J=6.78, 2H) and 2.32-2.23 (m, 2H).

[0165] Amino Nitro Acid (24)

[0166] A solution of potassium hydroxide (0. 5 g, 8.7 mmol) and thenitrobenzoic acid 23 (1 g, 2.9 mmol) in aqueous methanol (H₂O, 10 mL;methanol, 20 mL) was allowed to stir at room temperature for 1 hour andthen heated at reflux until TLC (AcOEt, MeOH, TEA, 1:10:100) revealedthe complete consumption of starting material. Excess methanol wasremoved by rotary evaporation and the residual solution diluted withwater and neutralised with IN HCl. The neutralised aqueous solution wasused directly, without further purification, in the next synthetic step.

[0167] Fmoc Nitro Acid (25)

[0168] Fluorenylmethyl chloroformate (0.78 g, 3 mmol) was addedportionwise to the aqueous solution from the previous reaction which hadbeen diluted with THF (50 mL) and aqueous sodium carbonate (2.15 g, 50mL water). The reaction mixture was then allowed to stir overnight.Excess organic solvent was. removed by rotary evaporation under reducedpressure from the reaction mixture, the residual aqueous solution wasthen washed with ethyl acetate (3×20 mL) (to remove excess Fmoc-Cl). Theaqueous phase was acidified with conc. HCl and extracted with ethylacetate (2×50 ML). The organic phase was dried over magnesium sulphate,filtered and evaporated in vacuo to afford the product 25 (1 g, 70%yield). ¹H NMR (270 MHz, CDCl₃) δ (Rotamers) 8.21 (bs, 2H), 7.73 (d,J=7.14 Hz, 2H), 7.59 (d, J=7.33 Hz, 2H) 7.40-7.13 (m, 5H), 6.47 and 5.70(2×bs, 1H), 4.54-3.88 (m, 5H), 3.77 (s, 3H), 3.44-3.42 (m, 2H) and2.04-1.90 (m, 2H). ^(—)C NMR (68.7 MHz, CDCl₃) δ 168.7, 156.9, 152.1,149.8, 143.7, 141.9, 141.3, 127.7, 127.0, 124.9, 120.6, 120.0, 111.1,107.8, 68.5, 66.4, 56.4, 47.3, 39.1 and 28.4.

[0169] Fmoc Nitro Alcohol (26)

[0170] A catalytic amount of DMF (2 drops) was added to a solution ofthe acid 25 (1.16 g, 2.36 mmol) and oxalyl chloride (0.33 g, 2.6 mmol)in dry dichloromethane (20 mL) and the reaction mixture was allowed tostir overnight. The resulting acid chloride solution was cooled to 0° C.and treated dropwise with a solution of pyrrolidinemethanol (0.26 g,2.57 mmol) and triethylamine (0.52 g, 5.14 mmol) in dry dichloromethane(15 mL). Thin layer chromatography, performed shortly after the end ofthe addition of amine, revealed that reaction had gone to completion.The reaction mixture was washed with HCl (lN, 1×50 mL) and water (2×20mL) and dried over magnesium sulphate. Removal of excess solventafforded the crude product which was subjected to flash columnchromatography (silica gel, gradient elution, 1% methanol in chloroformto 2% methanol in chloroform) to afford the required amide 26 (1.1 g,81%). ¹H NMR (270 MHz, CDCl₃) δ 7.75 (d, J=7.33 Hz, 2H), 7.67 (s, 1H),7.60 (d, J=6.96 Hz, 2H), 7.41-7.26 (m, 4H), 6.78 (s, 1H), 5.66 (bs, 1H),4.48-4.39 (m, 3H), 4.23-4.13 (m, 3H), 3.91-3.79 (m, 5H), 3.45-3.42 (m,2H), 3.18-3.13 (m, 2H) and 2.08-1.70 (m, 6H). ¹³C NMR (68.7 MHz, CDCl₃)δ 168.5, 156.5, 154.7, 148.2, 143.9, 141.3, 137.0, 128.0, 127.7, 127.0,124.9, 120, 108.9, 108.0, 68.4, 66.2, 66.0, 61.5, 56.6, 53.5, 47.3,39.0, 28.9, 28.4 and 24.4.

[0171] Fmoc Amino Alcohol (27)

[0172] A solution of the nitroamide 26 (3 g, 5.22 mmol) and SnCl₂ 2H₂O(6.15 g, 27.15 mmol) in methanol (60 mL) was heated at reflux for 2hours. The reaction mixture was concentrated to ⅓ of its original volumeand carefully treated with saturated aqueous sodium bicarbonate solution(vigorous effervescence!) until pH8 was obtained. The mixture wasallowed to stir vigorously with ethyl acetate (100 mL) overnight andthen filtered through celite to remove precipitated tin salts. Theaqueous phase was extracted with ethyl acetate (50 mL) and the combinedorganic phase was dried over magnesium sulphate. Removal of excesssolvent afforded the desired amine as a dark yellow oil 27 (1.93 g,68%). ¹H NMR (270 MHz, CDCl₃) δ 7.75 (d, J=7.51 Hz, 2H), 7.61 (d, J=7.33Hz, 2H), 7.40-7.26 (m, 4H), 6.72 (s, 1H), 6.25 (s, 1H), 5.95 (bs, 1H),4.43-4.04 (m, 6H), 3.67-3.42 (m, 9H) and 2.11-1.7 (m, 6H). ¹³C NMR (68.7MHz, CDCl₃) δ 171.7, 156.6, 150.8, 144.0, 141.3, 140.6, 127.6, 127.0,125.0, 119.9, 112.0, 102.2, 68.0, 66.6, 66.4, 61.0, 56.6, 51.0, 47.3,39.5, 29.1, 28.5 and 24.9.

[0173] Resin-Bound Amino-Alcohol (28)

[0174] The o-nitrobenzylchloroformate resin (0.048 mmol) was allowed toswell for 10 minutes in dry dichloromethane (5 mL). A solution of theamino alcohol (0.13 mg, 0.24 mmol) and pyridine (0.02 g) in drydichloromethane (1 mL) was added to the resin suspension under anitrogen atmosphere at 0° C. The reaction mixture was then allowed toshake overnight at room temperature. Excess reagent was removed bysuction and the resin washed with dichloromethane (2×5 mL) and methanol(2×5 mL) and then dried in vacuo overnight. The procedure was repeatedthree times to ensure complete reaction.

[0175] Resin-Bound Fmoc-Aminoproyl PBD (29)

[0176] The carbamate resin (0.048 mmol) prepared in the previousreaction was allowed to swell in dry dichloromethane (1 mL) for 10minutes. The suspension was cooled to −10° C. and treated successivelywith triethylamine (20 μL, 0.144 mmol) and pyridine sulphur trioxidecomplex (0.023 g, 0.144 mmol) in dimethyl sulphoxide (0.5 mL) at −10° C.and the suspension was allowed to shake at −10° C. for two hours. Excessreagent was removed by suction and the resin washed with methanol (2×5mL) and dichlorbmethane (2×5 mL) and then dried in vacuo overnight. Theprocedure was repeated three times to ensure-complete reaction.

[0177] Resin-Bound Aminopropyl PBD (30)

[0178] The resin-bound 8-aminopropyl PBD 30 is prepared from theFmoc-protected form 29 by standard deprotection conditions. Thiscompound 30 can be used to form compounds according to the second aspectof the present invention, by reaction of appropriate combinatorial unitsfrom those described above with this compound.

EXAMPLE 8 Synthesis of Resin Bound 8-Allyl Ester Protected Acid PBDScaffold (39) (see FIG. 10)

[0179] The resin bound PBD 39 was synthesised following the strategyused in Example 4.

[0180] Synthesis of Amine 37

[0181] The nitro acid 35 was derived from the alcohol 35a by thefollowing two steps.

[0182] The alcohol 35a (50 g, 0.22 mol) was added portionwise over 1hour to nitric acid (70%, 400 ml) cooled to 0° C. Once addition wascomplete, the solution was stirred at 0° C. for 1 hour, then allowed towarm to room temperature. The semisolid formed was collected byfiltration and washed with a minimum of ice/water. The resulting paleyellow solid was redissolved in EtOAc, the solution dried (MgSO₄) andthen concentrated to afford the diacid 35b (31 g, 49%). ¹H NMR (270MHZ): δ 2.83-2.79 (t, J=6, 12.5 Hz, 2H), 3.94 (s, 3H), 4.37-4.33 (t,J=6, 12.5 HZ, 2H), 7.18 (s, 1H), 7.46 (s, 1H), 10.38 (br.s, 2H).

[0183] A mixture of the diacid 35b (20 g, 74.3 mmol) and p-toluenesulphonic acid monohydrate (2.3 g, 7.4 mmol) in allyl alcohol (240 mL,3.5 mol) was refluxed for 7 hours then allowed to cool. The allylalcohol was then removed in vacuo, and the residue triturated withdilute HCl acid (3×75 ml) and collected by filtration. This solid wastaken up in EtOAc, and the resulting solution washed with water (3×50ml) and brine (3×50 ml) and dried over sodium sulphate. Evaporation invacuo afforded 35 as a white solid (19.27 g, 84%): mp 128-130° C.;¹H-NMR (270 MHZ, CDCl₃) δ 2.92 (t, 2H, J=6.35 Hz); 3.94 (s, 3H); 4.38(t, 2H, J=6.41 Hz); 4.65 (d, 2H, J=5.61 Hz); 5.27 (dd, 1H, J₁=1.28 Hz,J₂=19.42 Hz); 5.33 (dd, 1H, J₁=1.28 Hz, J₂=17.04 Hz); 5.92 (m, 1H); 7.15(s, 1H); 7.45 (s, 1H); ¹³C NMR (67.8 MHZ, CDCl₃): δ 34.1, 56.5, 65.0,65.4, 108.5, 111.3, 118.3, 122.9, 131.8, 141.1, 149.1, 152.6, 167.1,170.0; IR (Nujol); ν 1730, 1630, 1550, 1430, 1390, 1290, 1230, 1190,1170, 1070, 1030, 1010 cm⁻¹; MS (EI) m/z (relative intensity): 325 (M⁺,19), 251 (3), 213 (2), 196 (3), 211 (3), 113 (19), 91 (4), 71 (9), 55(6); HRMS: calcd. for. C₁₄H₁₅NO₈ 325.0798, found 232.0773.

[0184] To a suspension of the nitro acid 35 (5 g, 0.015 mol) in CH₂Cl₂(75 ml) was added oxalyl chloride (1.5 ml, 0.017 mol) and DMF (0.05 ml)in a dropwise manner and the resulting solution was stirred for 16hours. The acid chloride was then added dropwise to a stirring solutionof pyrrolidine methanol (1.7 g, 0.017 mol) and triethylamine (4.7 ml,0.034 mol) in CH₂Cl₂ (40 ml) at −20° C. (liquid N₂/acetone). Thissolution was stirred at room temperature for 16 hours. The reaction wasquenched with aqueous HCl (1.0 N, 25 ml) and the organic extracts werewashed with H₂O and brine, dried and concentrated to give the crudeyellow oil. The material was purified by column chromatography (5%MeOH/CHCl₃), to give 36 as a pale yellow oil (6.2 g, 100%). ¹HNMR (270MHz, CDCl₃) δ 2.22-1.71 (m, 6H); 2.94 (t, J=6.4 Hz, 2H); 3.15 (d×d,J=6.5 Hz, 2H); 3.92-3.76 (m, 1H); 3.96 (s, 3H); 4.4 (t, J=6.2 Hz, 2H);4.67-4.64 (m, 2H); 5.39-5.23 (m, 2H); 6.0-5.86 (m, 1H); 6.81 (s, 1H);7.75 (s, 1H) .

[0185] To a stirring solution of the nitro compound 36 (6 g, 0.015 mol)in MeOH (80 ml) was added SnCl₂.2H₂O (16.6 g, 0.074 mol) and heated atreflux for 45 minutes. The reaction was concentrated in vacuo and theresidual oil was partitioned between EtOAc and aqueous saturated NaHCO,and stirred vigorously for 16 hours to aid separation. This material wasfiltered through Celite and extracted with EtOAc, washed with H₂O andbrine, dried and concentrated to give amine 37 as a yellow oil (3.3 g,59%). ¹H NMR (270 MHz, CDCl₃) δ 2.17-1.65 (m, 6H); 2.9 (t, J=6.6 Hz,2H); 3.72-3.46 (m, 3H); 3.75 (s, 3H); 4.2 (t, J=6.8 Hz, 2H); 4.4 (br.d×d, J=9.7 Hz, 2H); 4.65-4.62 (m, 2H); 5.37-5.22 (m, 2H); 6.0-5.85 (m,1H); 6.3 (s, 1H); 6.76 (s, 1H).

[0186] Synthesis of PBD Scaffold 39

[0187] A suspension of p-nitrophenyl carbamate Wang resin 31 (1 g, 0.6mmol/g loading) in CH₂Cl₂/DMF (2:1, 15 ml) was shaken for 30 minutes. Asolution of amine 37 (1.13 g, 3 mmol), HOBt (0.24 g, 1.8 mmol) and DIPEA(0.63 ml, 3.6 mmol) in CH₂Cl₂/DMF (2:1, 15 ml) was added to the swollenresin. The vessel was allowed to shake at room temperature for 6 hours.Resin 38 was filtered and rinsed with DMF (2×10 ml), CH₂Cl₂ (2×10 ml),MeOH (2×10 ml), Et₂O (10 ml) and dried in vacuo.

[0188] A suspension of resin 38 (0.6 mmol) in CH₂Cl₂ (10 ml) was allowedto shake for 30 min. A solution of Dess Martin periodinane (1.27 g, 3mmol) in CH₂Cl₂ (20 ml) was added to the swollen resin. The vessel wasallowed to shake at room temperature for 2 hours. Resin 39 was filteredand rinsed with CH₂Cl₂ (2×10 ml), MeOH (2×10 ml), Et₂O (10 ml) and driedin vacuo.

[0189] This scaffold 39 could be used in combinatorial chemistry bydeprotection of the acid group using Pd(PPh,), in chloroform, aceticacid and n-methyl morpholine.

[0190] A suspension of resin 39 (0.6 mmol) in TFA/CH₂Cl₂ (1:1, 30 ml)was allowed to shake for 2 hours. The resultant red solution wasdecanted off and was diluted with water (20 ml) and carefullyneutralised to pH 7.0 by the addition of solid sodium bicarbonate. Theorganic phase was separated and washed with H₂O (3×20 ml), brine (2×20ml), dried (MgSO₄) and concentrated to give a brown foam 40 (0.09 g,42%). EIMS 358 (M+1)⁺

EXAMPLE 9 Synthesis of an Aminopropyloxy PBD Scaffold (50) (See FIG. 11)

[0191] Overall Synthesis Strategy

[0192] The on-bead (p-nitrophenyl Wang resin) ring closure (49-50) wascarried out using a Dess Martin reagent as in Example 4. The final step(production of the off-bead PBD), was carried out to prove that theon-bead resin was of the desired structure.

[0193] Synthesis of N-(tert-butoxycarbonyl)-3-hydroxypropylamine 42

[0194] A solution of (Boc)₂O (25.0 g, 114.5 mmol) in anhydrous DCM (50mL) was added dropwise to a stirred solution of 3-amino-1-propanol 41(7.8 g, 104.5 mmol) in anhydrous DCM (100 mL), under a nitrogenatmosphere. The reaction mixture was allowed to stir for 12 hours, afterwhich time TLC (50% pet-ether/EtOAc) revealed complete loss of startingmaterial. The solution was diluted with Et₂O (150 mL) and washed withphosphate buffer (0.5 M, pH 5.4, 2×70 mL), sat. aqueous NaHCO₃ (70 mL),brine (2×70 mL) and dried over MgSO₄. Excess solvent was removed byevaporation under reduced pressure to give a viscous colourless oil(18.3 g, 100%).

[0195]¹H NMR (270 MHz, CDCl₃): δ 1.44 (s, 9H, CH₃), 1.67 (m, 2H, H2′),3.26 (q, 2H, J=6.23 Hz, H3′), 3.65 (dd, 2H, J=5.86, 5.68 Hz, H1′), 3.78(dt, 1H, J=6.04, 5.87 Hz, OH), 5.18 (br, 1H, NH); ¹³C NMR (67.8 MHz,CDCl₃): δ 28.4 (CH₃), 32.6 (C2′), 37.1 (C3′), 59.3 (C1′), 79.4(C_(quater)), 157.1 (C═O); MS (E/I) m/z (relative intensity): 176 (M⁺,30), 120 (100), 119 (31), 102 (49), 83 (33), 76 (67), 74 (36); HRMS(E/I) exact mass calcd for C₈H₁₇O₃N: m/e 175.1200, obsd m/e 175.1208; IR(Nujol) ν: (cm⁻¹) 3355, 2976, 2936, 2878, 1810, 1694, 1531, 1455, 1392,1366, 1278, 1253, 1173, 1072, 996, 914, 870, 781, 752, 638.

[0196] Synthesis of Methyl4-[N-(tert-butoxycarbonyl)]aminopropyloxy-3-methoxybenzoate 44

[0197] A solution of DEAD (18.3 g, 105.3 mmol) in freshly distilled THF(50 ml) was added dropwise to a mechanically stirred solution oftriphenylphosphine (27.6 g, 105.3 mmol), methyl vanillate 43 (19.2 g,105.3 mmol), and Boc-amino-1-propanol 42 (18.4 g, 105.3 mmol) in freshlydistilled THF (250 mL), at 0° C. under a nitrogen atmosphere. Followingthe addition of DEAD the reaction mixture was allowed to stir at roomtemperature overnight and the progress of reaction was monitored by TLC(50% EtOAc/pet-ether). The solvent was removed by evaporation underreduced pressure and the residue was triturated with Et₂O (300 mL). Thisled to the precipitation of some of the TPO and diethylhydrazinedicarboxylate which were removed by filtration and the filtratewas washed with 1 N aqueous NaOH (150 mL), H₂O (2×150 mL), brine (2×150mL) and dried over MgSO₄. Excess solvent was removed by evaporationunder reduced pressure. The title compound was purified by columnchromatography (80% pet-ether/EtOAc) to give a beige solid (30 g, 85%).

[0198] mp=79-82° C.; ¹H-NMR (CDCl₃, 270 MHz): δ 1.46 (s, 9H, CH₃),2.0-2.08 (m, 2H, H2′), 3.38 (dd, 2H, J=5.68, 6.04 Hz H3′), 3.90 (s, 3H,OCH_(3ester)). 3.93 (s, 3H, OCH_(3ether)), 4.14 (t, 2H, J=5.95 Hz, H3′),5.58 (br, 1H, NH), 6.86 (d, 1H, J=8.42 Hz, H5), 7.55 (d, 1H, J=1.83 Hz,H2), 7.65 (dd, 1H, J=2.02, 8.42 Hz, H6); ¹³C-NMR (CDCl₃, 68.7 MHz): δ28.5 (C), 29.2 (C2′), 38.9 (C3′), 52.0 (OCH_(3ester)), 55.8(OCH_(3ether)), 68.1 (C1′), 78.9 (C_(quater)), 111.3 (C5), 112.0 (C2),122.84 (C_(arom)), 123.5 (C6), 148.8 (C_(arom)), 152.1 (C_(arom)), 156.1(NC═O), 166.8 (C═O); MS (E/I) m/z (relative intensity): 339 (M⁺, 11),266 (13), 182 (42), 151 (27), 102 (100); HRMS (E/I) exact mass calcd.for C₁₇H₂₅NO₆: m/e 339.1682, obsd m/e 339.1733; IR (Nujol) ν: (cm⁻¹)3362, 2923, 2854, 1712, 1684, 1599, 1520, 1464, 1377, 1272, 1217, 1132,1045, 1022, 872, 780, 762, 722.

[0199] Synthesis of Methyl 4-Aminoproyloxy-5-methoxy-2-nitrobenzoate 45

[0200] The ester 44 (4.0 g, 11.8 mmol) was added in small portions to astirred solution of 70% HNO₃ (2 mL acid/g of substrate) at roomtemperature and the reaction mixture was allowed to stir overnight. TLC(CHCl₃) at this point revealed the complete loss of starting material.The reaction mixture was cooled on ice bath, and 15 g of iced water wasadded, precipitating the product. The precipitate was collected byvacuum filtration and washed with small amount of iced water. Thefiltrate was cooled and a second crop of precipitate was collected byvacuum filtration and washed with iced water. The combined precipitatewas dried in vacuo to provide the title compound 45 as a yellow solid,which was not purified further, but used directly in the subsequentreaction (2.3 g, 70%).

[0201] mp=101-103° C.; ¹H-NMR (CDCl₃/DMSO-d₆, 270 MHz): δ 2.31 (m, 2H,H2′), 3.20 (br, 2H, H3′), 3.95 (s, 3H, OCH_(3ether)), 3.98 (s, 3H,OCH_(3ester)), 4.24 (t, 2H, J=5.95 Hz, H1′), 7.11 (s, 1H, H6), 7.49 (s,1H, H3), 8.21 (s, 3H, NH); ¹³C-NMR (CDCl₃, 68.7 MHz): δ 26.5 (C2′), 37.0(C3′), 53.0 (OCH_(3ester)), 56.0 (OCH_(3ether)), 66.7 (C1′), 108.3 (C3),111.0 (C6), 121.6 (C_(arom)), 140.9 (C2), 149.3 (C_(arom)), 152.6(C_(arom)), 166.8 (C═O); MS (E/I) m/z (relative intensity): 284 (M⁺,90), 237 (70), 227 (93), 196 (47), 181 (38), 137 (100), 122 (81), 93(52), 79 (44); HRMS (E/I) exact mass calcd. for C₁₂H₁₇N₂O₆: m/e284.1008, obsd m/e 284.1018; IR (Nujol) ν: (cm⁻¹) 3472, 2937, 2911,2855, 1733, 1532, 1516, 1462, 1377, 1292, 1224, 1143, 1052, 884, 812,792, 773, 756, 724, 646.

[0202] Synthesis of Methyl4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-nitrobenzoicacid 46

[0203] A solution of 45 (3.9 g, 11.2 mmol) and KOH (1.9 g, 33.4 mmol) inaqueous methanol (77 mL MeOH, 15 mL H₂O) was-heated at reflux for 90minutes. At which time TLC (EtOAc/MeOH/TEA 100:10:1) revealed completeconsumption of starting material. Excess MeOH was removed by evaporationunder reduced pressure and the concentrate diluted with H₂O (20 mL). Theaqueous solution was neutralised with conc. HCl, diluted with THF (100mL) and sodium carbonate (2.9 g, 27.9 mmol) was added to adjust thesolution to pH 9. After this, fluorenylmethyl chloroformate (3.0 g, 11.6mmol) was added portionwise over 30 minutes and the reaction mixture wasallowed to stir for 12 hours. Excess THF was removed by evaporationunder reduced pressure and the aqueous fraction was extracted with EtOAc(3×100 mL) to remove free Fmoc, and then acidified with conc. HCl andextracted again with EtOAc (3×100 mL). The organic phase was washed withH₂O (2×100 mL), brine (100 mL), dried over MgSO₄, and excess solvent wasremoved by evaporation under reduced pressure to afford 46 as a beigesolid which was not purified further, but used directly in thesubsequent reaction (4.7 g, 86%).

[0204] mp=145-146° C.; ¹H-NMR (CDCl₃, 270 MHz): δ 1.81 (m, 2H, H2′),3.43 (m, 2H, H3′), 3.78 (s, 3H, OCH₃), 4.08-4.23 (m, 3H, H1′+FMoc CH),4.49 (d, 2H, J=6.41 Hz, Fmoc CH₂), 5.70 (br, 1H, NH), 7.14 (s, 1H, H6),7.26-7.41 (m, 5H, Fmoc_(aryl)H3), 2 +H3), 7.59 (d, 2H, J=7.51 Hz,Fmoc_(aryl)), 7.74 (d, 2H, J=7.15 Hz, Fmoc_(aryl)), 9.62 (s, 1H, CO₂H);¹³C-NMR (CDCl₃, 68.7 MHz): δ 28.8 (C2′), 39.1 (C3′), 47.2 (CH Fmoc),56.4 (OCH₃)), 66.3 (CH₂ Fmoc), 68.5 (C1′), 107.9 (C3), 111.1 (C6),120.0, 124.9, 127.1 and 127.7 (CH Fmoc_(aryl)), 128.0 (C_(arom)), 137.0(C_(arom)), 141.3 (C Fmoc_(aryl)), 143.8 (C Fmoc_(aryl)); 148.2(C_(arom)), 154.7 (C_(arom)), 156.8 (NC═O) 171.5 (CO₂H); MS (FAB) m/z(relative intensity): 493 (M⁺+1, 3), 297 (6), 271 (4), 191 (18), 180(21), 179 (100), 178 (67), 165 (30), 102 (17), 93 (13); HRMS (FAB) exactmass calcd. for C₂₆H₂₅N₂O₈ (M+H): m/e 493.1532, obsd m/e 493.1536; IR(Nujol™) ν: (cm⁻¹) 1712, 1535, 1463, 1377, 1277, 1219, 1081, 970, 762,722, 656.

[0205] Synthesis of(2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-methanol47

[0206] A catalytic amount of DMF (2 drops) was added to a solution ofthe nitrobenzoic acid 46 (8.0 g, 16.3 mmol) and oxalyl chloride (2.3 g,17.9 mmol) in anhydrous DCM (120 mL), at room temperature under anitrogen atmosphere. The reaction mixture was stirred for 16 hours andthe resulting solution of acid chloride was cooled to 0° C.(ice/acetone) under a nitrogen atmosphere. A solution ofpyrrolidinemethanol (1.8 g, 17.9 mmol) and DIPEA (4.6 g, 35.77 mmol) inanhydrous DCM (40 mL) was added dropwise over 30 minutes. Once theaddition was complete, the reaction mixture was allowed to warm to roomtemperature. Stirring was continued for a further 2 hours, at which timeTLC (95% EtOAc/MeOH) revealed complete reaction. The reaction mixturewas washed with 1 N aqueous HCl (2×100 mL), H₂O (2×100 mL), brine (100mL), and dried over MgSO₄. Excess solvent was removed by evaporationunder reduced pressure to give the crude compound as a brown oil.Purification by flash column chromatography (99% CHCl₃/MeOH) affordedthe pure amide 47 as a beige solid (5.6 g, 82%)

[0207] [α]_(D)=−53.3° (c=1.03, CHCl₃); mp=78-81° C.; ¹H-NMR (CDCl₃, 270MHz): δ 1.69-1.88 (m, 4H, H4+H3), 2.04-2.12 (m, 2H, H2′), 3.16 (m, 2H,H3′), 3.45 (m, 2H, H5), 3.81 (s, 3H, OCH₃), 3.86-3.91 (m, 2H, CH₂—OH),4.08-4.24 (m, 3H, H1′+Fmoc CH), 4.38-4.48 (m, 3H, H2+Fmoc CH₂), 5.65(br, 1H, NH), 6.78 (s, 1H, H6_(arom)), 7.27-7.42 (m, 5H,H3_(arom)+Fmoc_(aryl)), 7.61 (d, 2H, J=7.32 Hz, Fmoc_(aryl)), 7.76 (d,2H, J=7.32 Hz, Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz): δ 24.4 (C4),28.4 (C3), 28.9 (C2′), 39.1 (C3′), 47.3 (CH Fmoc), 49.5 (C5), 56.6(OCH)), 60.4 (C2), 61.5 (CH₂—OH), 66.2 (CH₂ Fmoc), 68.5 (C1′), 108.0(C3_(arom)), 108.9 (C6_(arom)), 120.0, 124.9, 127.0 and 127.7 (CHFmoc_(aryl)), 128.0 (C_(arom)), 137.0 (C_(arom)), 141.3 (C Fmoc_(aryl)),143.9 (C Fmoc_(aryl)), 148.2 (C_(arom)), 154.7 (C_(arom)), 156.5(NC═O_(carbamate)), 171.2 (C═O_(amide)); MS (FAB) m/z (relativeintensity): 576 (M⁺+1, 32), 191 (18), 179 (100), 165 (25), 102 (33);HRMS (FAB) exact mass calcd for C₃₁H₃₄N₃O₈ (M+H): m/e 576.2268 obsd m/e576.225; IR (Nujol) ν: (cm⁻¹) 2626, 1714, 1615, 1576, 1520, 1452, 1434,1333, 1276, 1218, 1147, 1059, 869, 818, 759, 742.

[0208] Synthesis of (2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-aminobenzoyl]pyrrolidine-2-methanol 48

[0209] A mixture of the nitro compound 47 (5.5 g, 9.5 mmol) andSnCl₂/2H₂O (10.2 g, 45.4 mmol) in MeOH (100 mL) was heated at reflux andthe progress of the reaction monitoring by TLC (95% CHCl₃/MeOH). After 2hours excess MeOH was removed by evaporation under reduced pressure, theresulting residue was cooled (ice), and treated carefully with sat.aqueous NaHCO₃ (170 mL). The reaction mixture was diluted with EtOAc(170 mL) and after 16 hours stirring at room temperature the inorganicprecipitate was removed by filtration through Celite. The organic layerwas separated, washed with brine (150 mL), dried over MgSO₄, filteredand evaporated in vacuo to give a brown solid. Purification by flashcolumn chromatography (95% CHCl₃/MeOH) afforded the pure amine 48 as agreyish-pink solid (4.3 g, 82%)

[0210] [α]_(D)=−78.6° (c=1.02, CHCl₃); mp=83-86° C.; ¹H-NMR (CDCl₃, 270MHz): δ 1.68-1.85 (m, 4H, H4+H3), 2.00-2.04 (m, 2H, H2′), 3.43-3.45 (m,2H, H3′), 3.49-3.58 (m, 2H, H5), 3.67 (s, 3H, OCH₃), 3.72-3.78 (m, 2H,CH₂—OH), 4.04 (t, 2H, J=5.58 Hz, H1′), 4.22 (t, 1H, J=6.86 Hz, Fmoc CH),4.41-4.44 (m, 3H, H2+Fmoc CH₂), 5.92 (br, 1H, NH), 6.23 (s, 1H,H3_(arom)), 6.71 (s, 1H, H6_(arom)), 7.27-7.41 (m, 4H, Fmoc_(aryl)),7.62 (d, 2H, J=7.32 Hz, Fmoc_(aryl)), 7.75 (d, 2H, J=7.33 Hz,Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz): δ 24.9 (C4), 28.6 (C3), 29.1(C2′), 39.5 (C3′), 47.3 (CH Fmoc), 51.0 (C5), 56.6 (OCH₃), 60.4 (C2),61.1 (CH₂—OH), 66.4 (CH₂ Fmoc), 68.0 (C1′), 102.0 (C3_(arom)), 111.6(C6_(arom)), 120.0, 125.1, 127.0 and 127.7 (CH Fmoc_(aryl)), 128.0(C_(arom)), 137.8 (C_(arom)), 141.3 (C Fmoc_(aryl)), 144.0 (CFmoc_(aryl)), 148.2 (C_(arom)), 150.8 (C_(arom)), 156.6(NC═O_(carbamate)), 171.9 (C═O_(amide)); MS (FAB) m/z (relativeintensity): 546 (M⁺1, 11), 445 (10), 191 (14), 179 (100), 166 (51), 102(70); HRMS (FAB) exact mass calcd for C₃₂H₃₇N₃O₆ (M+H): m/e 546.2526obsd m/e 546.2532; IR (Nujol) ν: (cm⁻¹) 1698, 1622, 1588, 1506, 1476,1404, 1228, 1173.

[0211] Synthesis of (2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-(N-resin-methoxybenzyloxycarbonyl)aminobenzoyl]pyrrolidine-2-methanol49

[0212] The p-nitrophenyl carbonate Wang resin 31 (1 g, 0.54 mmol) wasallowed to swell for 30 minutes in DCM/DMF (2:1, 10 mL) with gentleshaking in a round bottom flask equipped with a sintered glass filtertube. A solution of HOBt (0.22 g, 1.6 mmol), DIPEA (0.56 mL, 0.42 g, 3.2mmol) and the amine 48 (1.47 g, 2.7 mmol) in DCM/DMF (2:1, 20 mL) wasadded to the swollen resin. The reaction mixture was shaken for 6 hoursat room temperature and allowed to stand overnight, after this time thesupernatant was removed by vacuum filtration using the sintered filtertube. The resin was washed four times with DCM, MeOH, and Et₂O for 2minutes each. After this, the resin was dried in vacuo to afford theresin bound carbamate 49.

[0213] Synthesis of (11S,11aS)-10-(N-resin-methoxybenzyloxycarbamate)-11-hydroxy-8-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-7-methoxy-1,2,3,6,9,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one50

[0214] The carbamate bearing resin 49 (0.54 mmol) was allowed to swellfor 30 minutes in DCM/DMF (2:1, 10 mL) with gentle shaking. A solutionof the Dess-Martin reagent (1.14 9, 2.7 mmol) in DCM/DMF (2:1, 20 mL)was added to the swollen resin. The reaction mixture was shaken for 2hours at room temperature, after this time the supernatant was removedby vacuum filtration using the filter tube. The resin was washed fourtimes with DCM, MeOH, and Et₂O for 2 minutes each. The resin was driedin vacuo to afford the resin bound carbinolamine 50.

[0215] Synthesis of (11aS)8-(N-9-fluorenylmethoxycarbonyl)aminoproyloxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one51

[0216] The protected carbinolamine bound resin 50 (0.54 mmol) wassuspended in TFA/DCM (1:1, 20 mL) and shaken for 2 hours at roomtemperature. After this time the supernatant was decanted by pressureand the resin was washed twice with DCM. The deprotection protocol wasrepeated once to ensure complete cleavage of the PBD from the resin. Thecombined organic solution was diluted with water and carefullyneutralised with sat. aqueous NaHCO₃. The organic layer was separated,washed with H₂O (2×60 mL), brine (2×60 mL), dried over MgSO₄ and excessof solvent was removed by evaporation under reduced pressure.Purification by flash column chromatography (97% CHCl₃/MeOH) furnishedthe target compound 51 as a brown solid (67 mg, 24%) which wasrepeatedly evaporated in vacuo with CHCl₃ in an attempt to provide theN10-C11 imine form of the compound.

[0217] [α]_(D)=+397.5° (c=0.67, CHCl₃); ¹H-NMR (CDCl₃, 270 MHz): δ2.00-2.06 (m, 4H, H2+H1), 2.26-2.31 (m, 2H, H2′), 3.45-3.47 (m, 2H,H3′), 3.52-3.62 (m, 2H, H3), 3.80 (s, 3H, OCH₃), 3.91-4.24 (m, 4H,H11a+H1′+Fmoc CH), 4.43-4.46 (m, 2H, Fmoc CH₂), 5.93 (br, 1H, NH), 6.78(s, 1H, H6), 7.26-7.41 (m, 4H, Fmoc_(aryl)), 7.5 (s, 1H, H9), 7.61 (d,2H, J=7.14 Hz, Fmoc_(aryl)), 7.66 (d, 1H, J=4.39 Hz, H11_(imine)), 7.75(d, 2H, J=7.33 Hz, Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz): δ 24.2 (C2),29.0 (C1), 29.6 (C2′), 39.5 (C3′), 46.7 (C3), 47.4 (CH Fmoc), 53.7(OCH₃), 56.0 (C11a), 66.3 (CH₂ Fmoc), 68.2 (C1′), 110.3 (C6), 111.4(C9), 120.0 (C—H_(aryl) Fmoc), 120.5 (C_(arom)), 125.1, 127.0, and 127.6(C—H_(aryl) Fmoc), 140.6 (C_(arom)), 141.3 (C_(aryl) Fmoc), 144.0(C_(aryl) Fmoc), 147.7 (C_(arom)), 150.4 (C_(arom)), 156.6(NC═O_(carbamate)), 162.6 (C11), 164.5 (C4_(amide)); IR (Nujol) ν:(cm⁻¹) 3364, 1711, 1686, 1600, 1521, 1472, 1244, .1217, 1021, 740, 679.

EXAMPLE 10 Synthesis of 27 Member Tripeptide PBD Library 60 (FIG. 12)

[0218] A suspension of the amino PBD scaffold resin 30 (example 9) (0.45mmol), in 2:1 DCE:DMF (30 mL) was evenly distributed between 27 Alltechtubes (1.5 mL volume). The process was repeated twice, excess solventwas removed by suction and the resin was rinsed with CH₂Cl₂ (2×5 mL) anddried in vacuo.

[0219] A solution of Fmoc-amino acid (0.05 mmol/tube) [Fmoc-alanine 15mg/tube, Fmoc-glycine 14 mg/tube, Fmoc-phenylalanine 19 mg/tube], TBTU(15 mg, 0.05 mmol/tube) and DIPEA (8 mL, 0.05 mmol/tube) in DMF (500 mL)was added to resin 30 (0.017 mmol/tube) and allowed to shake for 16hours. Resin 55 was filtered and rinsed with DMF (2×1 mL), CH₂Cl₂ (2×1mL), MeOH (2×1 mL) and dried in vacuo.

[0220] The procedure was repeated once.

[0221] A solution of 20% piperidine in DMF (250 mL) was added to eachtube and shaken for 2 hours. Resin 56 was filtered and rinsed with DMF(2×1 mL), CH₂Cl₂ (2×1 mL), MeOH (2×1 mL) and dried in vacuo. Theprocedure was repeated once.

[0222] The above coupling and deprotection protocols were repeated twiceuntil the library of 27 tripeptides 60 was generated.

1-25. Cancelled.
 26. A compound of formula (I):

wherein: one of R₂, R₃, R₆, R₇ and R₈ is X—Y—A—, where X is selected from —COZ′, NHZ, SH, or OH, where Z is either H or an nitrogen protecting group, Z′ is either OH or an acid protecting group, Y is a divalent group such that HY═R, and A is O, S, NH, or a single bond; R₂ and R₃ (if not X—Y—A—) are independently selected from: H, R, OH, OR, ═O, ═CH—R, ═CH₂, CH₂—CO₂R, CH₂—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR and CN, and there is optionally a double bond between C1 and C2 or C2 and C3; R₆, R₇, R₈ and R₉ (if not X—Y—A—) are independently selected from H, R, OH, OR, halo, nitro, amino, Me₃Sn; or R₇ and R₈ together form a group —O—(CH₂)_(p)—O—, where p is 1 or 2; R₁₁ is either H or R; Q is S, O or NH; L is a linking group, or a single bond; O is a solid support; where R is a lower alkyl group having 1 to 10 carbon atoms, or an aralkyl group of up to 12 carbon atoms, whereof the alkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system, or an aryl group of up to 12 carbon atoms; and is optionally substituted by one or more halo, hydroxy, amino, or nitro groups, and optionally contains one or more hetero atoms, which may form part of, or be, a functional group.
 27. A compound according to claim 26, wherein it is either R₂ and/or R₈ that is X—Y—A—.
 28. A compound according to claim 26, wherein R, and HY if Y is present, are independently selected from lower alkyl group having 1 to 10 carbon atoms, or an alkaryl group of up to 12 carbon atoms, or an aryl group of up to 12 carbon atoms, optionally substituted by one or more halo, hydroxy, amino, or nitro groups.
 29. A compound according to claim 28, wherein R, and HY, if Y is present, are independently selected from lower alkyl group having 1 to 10 carbon atoms optionally substituted by one or more halo, hydroxy, amino, or nitro groups.
 30. A compound according to claim 29, wherein R, and HY, if Y is present, are unsubstituted straight or branched chain alkyl groups, having 1 to 10 carbon atoms.
 31. A compound according to claim 26, wherein Q is O.
 32. A compound according to claim 26, wherein R₁₁ is H.
 33. A compound according to claim 26, wherein R₆ and R₉ are H.
 34. A compound according to claim 26, wherein R₇ is an alkoxy group.
 35. A compound according to claim 26, wherein R₂ and R₃ are H.
 36. A collection of compounds all of which are represented by formula (I):

wherein one of R₂, R₃, R₆, R₇ and R₈ is H—(T)_(n)—X′—Y—A—, where: X′ is CO, NH, S or O,; Y is a divalent group such that HY═R; A is O, S, NH or a single bond; T is a combinatorial unit; and n is a positive integer. R₂ and R₃ (if not H—(T)_(n)—X′—Y—A—) are independently selected from: H, R, OH, OR, ═O, ═CH—R, ═CH₂, CH₂—CO₂R, CH₂—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR and CN, and there is optionally a double bond between C1 and C2 or C2 and C3; R₆, R₇, R₈ and R₉ (if not H—(T)_(n)—X′—Y—A—) are independently selected from H, R, OH, OR, halo, nitro, amino, Me₃Sn; or R₇ and R₈ together form a group —O—(CH₂)_(p)—O—, where p is 1 or 2; R₁₁ is either H or R; Q is S, O or NH; L is a linking group, or a single bond; O is a solid support; where R is a lower alkyl group having I to 10 carbon atoms, or an aralkyl group of up to 12 carbon atoms, whereof the alkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system, or an aryl group of up to 12 carbon atoms; and is optionally substituted by one or more halo, hydroxy, amino, or nitro groups, and optionally contains one or more hetero atoms, which may form part of, or be, a functional group.
 37. A collection of compounds according to claim 36, wherein it is R₂ and/or R₈ that are independently: H—(T)_(n)—X′—Y—A—.
 38. A collection of compounds according to claim 36, wherein X′ is either CO or NH.
 39. A collection of compounds according to claim 36, wherein n is from 1 to
 16. 40. A collection of compounds according to claim 39, wherein n is from 3 to
 14. 41. A collection of compounds all of which are represented by formula (II):

wherein one of R₂, R₃, R₆, R₇ and R₈ is H—(T)_(n)—X′—Y—A—, where: X′ is CO, NH, S or O,; Y is a divalent group such that HY═R; A is O, S, NH or a single bond; T is a combinatorial unit; and n is a positive integer. R₂ and R₃ (if not H—(T)_(n)—X′—Y—A—) are independently selected from: H, R, OH, OR, ═O, ═CH—R, ═CH₂, CH₂—CO₂R, CH₂—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR and CN, and there is optionally a double bond between C1 and C2 or C2 and C3; R₆, R₇, R₈ and R₉ (if not H—(T)_(n)—X′—Y—A—) are independently selected from H, R, OH, OR, halo, nitro, amino, Me₃Sn; or R₇ and R₈ together form a group —O—(CH₂)_(p)—O—, where p is 1 or 2; where R is a lower alkyl group having 1 to 10 carbon atoms, or an aralkyl group of up to 12 carbon atoms, whereof the alkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system, or an aryl group of up to 12 carbon atoms; and is optionally substituted by one or more halo, hydroxy, amino, or nitro groups, and optionally contains one or more hetero atoms, which may form part of, or be, a functional group.
 42. A collection of compounds according to claim 41, wherein it is R₂ and/or R₈ that are independently: H—(T)_(n)—X′—Y—A—.
 43. A collection of compounds according to claim 41, wherein X′ is either CO or NH.
 44. A collection of compounds according to claim 41, wherein n is from 1 to
 16. 45. A collection of compounds according to claim 44, wherein n is from 3 to
 14. 46. A method of screening a collection of compounds according to claim 41 to discover biologically active compounds. 